Antisense modulation of protein phosphatase 2 Catalytic subunit alpha expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of Protein Phosphatase 2 catalytic subunit alpha. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding Protein Phosphatase 2 catalytic subunit alpha. Methods of using these compounds for modulation of Protein Phosphatase 2 catalytic subunit alpha expression and for treatment of diseases associated with expression of Protein Phosphatase 2 catalytic subunit alpha are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of Protein Phosphatase 2 catalytic subunit alpha. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Protein Phosphatase 2 catalytic subunit alpha. Such compounds have been shown to modulate the expression of Protein Phosphatase 2 catalytic subunit alpha.

BACKGROUND OF THE INVENTION

[0002] The process of phosphorylation, defined as the attachment of a phosphate moiety to a biological molecule through the action of enzymes called kinases, represents one course by which intracellular signals are propagated resulting finally in a cellular response. Within the cell, proteins can be phosphorylated on serine, threonine or tyrosine residues and the extent of phosphorylation is regulated by the opposing action of phosphatases, which remove the phosphate moieties. While the majority of protein phosphorylation within the cell is on serine and threonine residues (Wera and Hemings, Biochemistry Journal, 1995, 311, 17-29), tyrosine phosphorylation is modulated to the greatest extent during oncogenic transformation and growth factor stimulation (Zhang, Crit. Rev. Biochem. Mol. Biol., 1998, 33, 1-52).

[0003] Because phosphorylation is such a ubiquitous process within cells and because cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or disorders are a result of either aberrant activation of, or functional mutations in, kinases and phosphatases. Consequently, considerable attention has been devoted recently to the characterization of these enzymes.

[0004] The enzyme protein phosphatase 2A (also known as PPP2A and PP2A) is one of four major protein phosphatases identified in the cytosol of eukaryotic cells which are responsible for the dephosphorylation of serine and threonine residues in proteins. These four enzymes have overlapping substrate specificities and are distinguished by their regulation and dependence on metal ions. Protein phosphatase 2A activity is independent of metal ions and appears to play a role in the regulation of major metabolic pathways, as well as the processes of translation, transcription, platelet activation and control of the cell cycle (Goldberg, Biochem. Pharmacol., 1999, 57, 321-328; Millward et al., Trends Biochem. Sci., 1999, 24, 186-191; Toyoda et al., Thromb. Haemost., 1996, 76, 1053-1062). More specifically, Protein Phosphatase 2A participates as a negative regulator in many kinase signal transduction pathways, including those involving MAP kinase, JNK kinase, ERK kinase, CaM kinase, and casein kinase. In addition, Protein Phosphatase 2A also interacts with many cellular and viral proteins (Millward et al., Trends Biochem. Sci., 1999, 24, 186-191). The enzyme has been shown to be activated by ceramide, a metabolic product of sphingomyelin hydrolysis and mediator of the biological effects of hormones, cytokines and growth factors (Dobrowsky et al., J. Biol. Chem., 1993, 268, 15523-15530).

[0005] The mammalian protein phosphatase 2A enzyme is a heterotrimer composed of a catalytic subunit of 36 kD complexed to two regulatory subunits, one of mass 65 kD and one of variable mass. In addition, two isoforms of the catalytic subunit of protein phosphatase 2A, alpha and beta, are demonstrable in many species. The structures of these catalytic subunits show high evolutionary conservation supporting the idea that they may serve crucial functions (Goldberg, Biochem. Pharmacol., 1999, 57, 321-328; Millward et al., Trends Biochem. Sci., 1999, 24, 186-191).

[0006] Protein Phosphatase 2 catalytic subunit alpha (also known as PPP2CA) was originally isolated from lung and lung fibroblast libraries (Stone et al., Nucleic Acids Res., 1988, 16, 11365), while the gene was isolated from a human leukocyte library (Khew-Goodall et al., Biochemistry, 1991, 30, 89-97). Northern analysis has revealed that the alpha subunit is expressed at relatively high levels compared to the beta subunit in all tissues examined. The structural characterization of the two genes implies that this is due in part to the different strengths of the promoters (Khew-Goodall et al., Biochemistry, 1991, 30, 89-97).

[0007] The catalytic subunit of Protein Phosphatase 2A has been linked to both insulin signaling (Klarlund et al., J. Biol. Chem., 1991, 266, 4052-4055; Kowluru et al., Endocrinology, 1996, 137, 2315-2323) and to retinoic acid-induced cellular differentiation of HL-60 cells, an acute promyelocytic leukemia cell line (Nishikawa et al., Cancer Res., 1994, 54, 4879-4884; Tawara et al., FEBS Lett., 1993, 321, 224-228). The pharmacological modulation of the catalytic subunit of Protein Phosphatase 2A activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions such as diabetes and cancer.

[0008] Currently, there are no known therapeutic agents which effectively inhibit the synthesis of the alpha isoform of Protein Phosphatase 2A catalytic subunit, and to date, investigative strategies aimed at modulating activity of Protein Phosphatase 2A function have involved the use of antibodies, molecules that block upstream entities, chemical inhibitors and gene knock-outs in mice.

[0009] Disclosed in U.S. Pat. Nos. 5,925,660 and 5,700,821 are compounds useful as phosphatase inhibitors and methods of making such inhibitors (Lazo et al., 1999; Lazo et al., 1997). It has also been reported that the compound, Fostriecin and compounds structurally related to it are effective serine/threonine phosphatase inhibitors. These are disclosed in the PCT publication WO 98/14606 (Honkanen and Downey, 1998). Disclosed in the PCT publication WO 99/27134 are antisense oligonucleotides targeting serine/threonine phosphatases, PP5, PP4 and PPlγl none of which target or hybridize to the Protein Phosphatase 2A isoforms (Honkanen and Dean, 1999).

[0010] In addition, at the protein level, there are compounds that interact with and consequently modulate the activity of the Protein Phosphatase 2A enzyme. These compounds and methods to identify these compounds are disclosed in the PCT publication WO 97/37037 (Hemmings, 1997).

[0011] Finally, homozygous null mutant mice are embryonically lethal, demonstrating that the alpha subunit gene is an essential gene (Gotz et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 12370-12375).

[0012] These strategies are untested as therapeutic protocols and consequently there remains a long felt need for additional agents capable of effectively inhibiting Protein Phosphatase 2A catalytic subunit alpha function.

[0013] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of Protein Phosphatase 2A catalytic subunit alpha expression.

[0014] The present invention provides compositions and methods for modulating Protein Phosphatase 2A catalytic subunit alpha expression.

SUMMARY OF THE INVENTION

[0015] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding Protein Phosphatase 2 catalytic subunit alpha, and which modulate the expression of Protein Phosphatase 2 catalytic subunit alpha. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of Protein Phosphatase 2 catalytic subunit alpha in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of Protein Phosphatase 2 catalytic subunit alpha by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding Protein Phosphatase 2 catalytic subunit alpha, ultimately modulating the amount of Protein Phosphatase 2 catalytic subunit alpha produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Protein Phosphatase 2 catalytic subunit alpha. As used herein, the terms “target nucleic acid” and “nucleic acid encoding Protein Phosphatase 2 catalytic subunit alpha” encompass DNA encoding Protein Phosphatase 2 catalytic subunit alpha, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of Protein Phosphatase 2 catalytic subunit alpha. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.

[0017] It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding Protein Phosphatase 2 catalytic subunit alpha. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding Protein Phosphatase 2 catalytic subunit alpha, regardless of the sequence(s) of such codons.

[0018] It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5¹-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon.

[0019] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.

[0020] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

[0021] Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0022] In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

[0023] Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target are identified through experimentation, and the sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The target sites to which these preferred sequences are complementary are hereinbelow referred to as “active sites” and are therefore preferred sites for targeting. Therefore another embodiment of the invention encompasses compounds which hybridize to these active sites.

[0024] Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.

[0025] For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0026] Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0027] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0028] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.

[0029] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[0030] While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 50 nucleobases (i.e. from about 8 to about 50 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.

[0031] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0032] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0033] Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0034] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0035] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0036] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0037] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0038] Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0039] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃; SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O-CH₂CH₂0CH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

[0040] A further prefered modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0041] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety. Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyl-adenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0042] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0043] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include inter-calators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0044] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0045] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0046] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0047] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0048] The antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules.

[0049] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0050] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0051] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0052] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

[0053] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.

[0054] For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

[0055] The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Protein Phosphatase 2 catalytic subunit alpha is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.

[0056] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding Protein Phosphatase 2 catalytic subunit alpha, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding Protein Phosphatase 2 catalytic subunit alpha can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of Protein Phosphatase 2 catalytic subunit alpha in a sample may also be prepared.

[0057] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

[0058] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.

[0059] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Prefered bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,. Prefered fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also prefered are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly prefered combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673 (filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624 (filed May 21, 1998) and 09/315,298 (filed May 20, 1999) each of which is incorporated herein by reference in their entirety.

[0060] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0061] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0062] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0063] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0064] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

Emulsions

[0065] The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.

[0066] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0067] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

[0068] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[0069] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0070] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[0071] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0072] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

[0073] In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0074] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[0075] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

[0076] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci. , 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

[0077] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

Liposomes

[0078] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

[0079] Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

[0080] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

[0081] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0082] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes. As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

[0083] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

[0084] Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.

[0085] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0086] Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0087] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0088] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0089] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[0090] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765). Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.)

[0091] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C₁₂15G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[0092] A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.

[0093] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0094] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0095] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0096] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0097] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0098] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0099] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

[0100] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[0101] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

[0102] Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0103] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0104] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0105] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0106] Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0107] Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.

[0108] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

[0109] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

[0110] In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0111] Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0112] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

[0113] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like. Other Components The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0114] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0115] Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0116] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0117] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0118] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy amidites

[0119] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2′-alkoxy amidites, the standard cycle for unmodified oligonucleotides was utilized, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds.

[0120] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C) nucleotides were synthesized according to published methods [Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

2′-Fluoro amidites 2′-Fluorodeoxyadenosine amidites

[0121] 2′-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a 2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and 5′ -DMT-3 ′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

[0122] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyrylarabinofuranosylguanosine. Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

[0123] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a literature procedure in which 2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-Fluorodeoxycytidine

[0124] 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-O-(2-Methoxyethyl) Modified amidites

[0125] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared as follows, or alternatively, as per the methods of Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.

2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0126] 5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid that was crushed to a light tan powder (57 g, 85% crude yield). The NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca. 5%). The material was used as is for further reactions (or it can be purified further by column chromatography using a gradient of methanol in ethyl acetate (10-25%) to give a white solid, mp 222-4° C.).

2′-O-Methoxyethyl-5-methyluridine

[0127] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 ml) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product. Additional material was obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0128] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0129] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by TLC by first quenching the TLC sample with the addition of MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl₃. The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%). An additional 1.5 g was recovered from later fractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0130] A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L), cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃ was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the latter solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0131] A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH₃ gas was added and the vessel heated to 100° C. for 2 hours (TLC showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0132] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, TLC showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0133] N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxytetra(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (TLC showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH₂Cl₂ (300 ML), and the extracts were combined, dried over MgSO₄ and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.

2′-O-(Aminooxyethyl)nucleoside amidites and 2′-O-(dimethylaminooxyethyl)nucleoside amidites 2′-(Dimethylaminooxyethoxy)nucleoside amidites

[0134] 2′-(Dimethylaminooxyethoxy)nucleoside amidites [also known in the art as 2′-O-(dimethylaminooxyethyl)nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0135] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) and the solution was cooled to −10° C. The resulting crystalline product was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMR were consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0136] In a 2 L stainless steel, unstirred pressure reactor was added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and with manual stirring, ethylene glycol (350 mL, excess) was added cautiously at first until the evolution of hydrogen gas subsided. 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160° C. was reached and then maintained for 16 h (pressure<100 psig). The reaction vessel was cooled to ambient and opened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T side product, ethyl acetate) indicated about 70% conversion to the product. In order to avoid additional side product formation, the reaction was stopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. [Alternatively, once the low boiling solvent is gone, the remaining solution can be partitioned between ethyl acetate and water. The product will be in the organic phase.] The residue was purified by column chromatography (2 kg silica gel, ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, stripped and dried to product as a white crisp foam (84 g, 50%), contaminated starting material (17.4 g) and pure reusable starting material 20 g. The yield based on starting material less pure recovered starting material was 58%. TLC and NMR were consistent with 99% pure product.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0137] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried over P₂O₅ under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture. The rate of addition is maintained such that resulting deep red coloration is just discharged before adding the next drop. After the addition was complete, the reaction was stirred for 4 hrs. By that time TLC showed the completion of the reaction (ethylacetate:hexane, 60:40). The solvent was evaporated in vacuum. Residue obtained was placed on a flash column and eluted with ethyl acetate:hexane (60:40), to get 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0138] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate was washed with ice cold CH₂Cl₂ and the combined organic phase was washed with water, brine and dried over anhydrous Na₂SO₄. The solution was concentrated to get 2′-O-(aminooxyethyl)thymidine, which was then dissolved in MeOH (67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was strirred for 1 h. Solvent was removed under vacuum; residue chromatographed to get 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).

[0139] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0140] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at 10° C. under inert atmosphere. The reaction mixture was stirred for 10 minutes at 10° C. After that the reaction vessel was removed from the ice bath and stirred at room temperature for 2 h, the reaction monitored by TLC (5% MeOH in CH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extracted with ethyl acetate (2×20 mL). Ethyl acetate phase was dried over anhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes. Reaction mixture cooled to 10° C. in an ice bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction mixture stirred at 10° C. for 10 minutes. After 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature for 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was added and extracted with ethyl acetate (2×25 mL). Ethyl acetate layer was dried over anhydrous Na₂SO₄ and evaporated to dryness. The residue obtained was purified by flash column chromatography and eluted with 5% MeOH in CH₂Cl₂ to get 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0141] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). This mixture of triethylamine-2HF was then added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reaction was monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed under vacuum and the residue placed on a flash column and eluted with 10% MeOH in CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0142] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P₂O₅ under high vacuum overnight at 40° C. It was then co-evaporated with anhydrous pyridine (20 mL). The residue obtained was dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the mixture and the reaction mixture was stirred at room temperature until all of the starting material disappeared. Pyridine was removed under vacuum and the residue chromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a few drops of pyridine) to get 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0143] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and dried over P₂O₅ under high vacuum overnight at 40° C. Then the reaction mixture was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 hrs under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated, then the residue was dissolved in ethyl acetate (70 mL) and washed with 5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrous Na₂SO₄ and concentrated. Residue obtained was chromatographed (ethyl acetate as eluent) to get 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy)nucleoside amidites

[0144] 2′-(Aminooxyethoxy)nucleoside amidites [also known in the art as 2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0145] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering A G (Berlin) to provide 2′-O-(2-ethylacetyl)diaminopurine riboside along with a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

[0146] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or 2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

[0147] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the solid dissolves. O²—,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oil bath and heated to 155° C. for 26 hours. The bomb is cooled to room temperature and opened. The crude solution is concentrated and the residue partitioned between water (200 mL) and hexanes (200 mL). The excess phenol is extracted into the hexane layer. The aqueous layer is extracted with ethyl acetate (3×200 mL) and the combined organic layers are washed once with water, dried over anhydrous sodium sulfate and concentrated. The residue is columned on silica gel using methanol/methylene chloride 1:20 (which has 2% triethylamine) as the eluent. As the column fractions are concentrated a colorless solid forms which is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine

[0148] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reaction mixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃ solution, followed by saturated NaCl solution and dried over anhydrous sodium sulfate. Evaporation of the solvent followed by silica gel chromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine) gives the title compound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0149] Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are added to a solution of 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere of argon. The reaction mixture is stirred overnight and the solvent evaporated. The resulting residue is purified by silica gel flash column chromatography with ethyl acetate as the eluent to give the title compound.

Example 2 Oligonucleotide Synthesis

[0150] Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine.

[0151] Phosphorothioates (P═S) are synthesized as for the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation wait step was increased to 68 sec and was followed by the capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (18 h), the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0152] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0153] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0154] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0155] Alkyl phosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0156] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0157] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0158] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3 Oligonucleoside Synthesis

[0159] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0160] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0161] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

[0162] Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.

Example 5 Synthesis of Chimeric Oligonucleotides

[0163] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides

[0164] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 380B, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for RNA and twice for 2′-O-methyl. The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 ammonia/ethanol at room temperature overnight then lyophilized to dryness. Treatment in methanolic ammonia for 24 hrs at room temperature is then done to deprotect all bases and sample was again lyophilized to dryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at room temperature to deprotect the 2′ positions. The reaction is then quenched with 1M TEAA and the sample is then reduced to 1/2 volume by rotovac before being desalted on a G25 size exclusion column. The oligo recovered is then analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0165] [2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxy-ethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl)Phosphodiester] Chimeric Oligonucleotides

[0166] [2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxy phosphorothioate]-[2′-O-(methoxyethyl)phosphodiester]chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidization with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0167] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

[0168] After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by ³¹P nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

[0169] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 96 well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per known literature or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0170] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8 Oligonucleotide Analysis—96 Well Plate Format

[0171] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96 well format (Beckman P/AC™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

[0172] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following 5 cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, Ribonuclease protection assays, or RT-PCR.

T-24 Cells

[0173] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0174] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

A549 Cells

[0175] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

NHDF Cells

[0176] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

HEK Cells

[0177] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recomnended by the supplier.

A10 Cells

[0178] The rat aortic smooth muscle cell line A10 was obtained from the American Type Culure Collection (Manassas, Va.). A10 cells were routinely cultured in DMEM, high glucose (American Type Culure Collection, Manassas, Va.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 80% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 2500 cells/well for use in RT-PCR analysis.

[0179] For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

Treatment with Antisense Compounds

[0180] When cells reached 80% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTI™ (Gibco BRL) and the desired concentration of oligonucleotide. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0181] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is ISIS 13920 TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to human H-ras. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments.

Example 10 Analysis of Oligonucleotide Inhibition of Protein Phosphatase 2 Catalytic Subunit Alpha Expression

[0182] Antisense modulation of Protein Phosphatase 2 catalytic subunit alpha expression can be assayed in a variety of ways known in the art. For example, Protein Phosphatase 2 catalytic subunit alpha mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0183] Protein levels of Protein Phosphatase 2 catalytic subunit alpha can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated activated cell sorting (FACS). Antibodies directed to Protein Phosphatase 2 catalytic subunit alpha can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

[0184] Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11 Poly(A)+ mRNA Isolation

[0185] Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C. was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0186] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

[0187] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY ₉₆™ plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water.

[0188] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13 Real-Time Quantitative PCR Analysis of Protein Phosphatase 2 Catalytic Subunit Alpha mRNA Levels

[0189] Quantitation of Protein Phosphatase 2 catalytic subunit alpha mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE, FAM, or VIC, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0190] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0191] PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 100 μM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 μL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0192] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreen are taught in Jones, L. J., et al, Analytical Biochemistry, 1998, 265, 368-374.

[0193] In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 25 uL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.

[0194] Probes and primers to human Protein Phosphatase 2 catalytic subunit alpha were designed to hybridize to a human Protein Phosphatase 2 catalytic subunit alpha sequence, using published sequence information (GenBank accession number M60483, incorporated herein as SEQ ID NO:3). For human Protein Phosphatase 2 catalytic subunit alpha the PCR primers were:

[0195] forward primer: CACTGGATCATATCAGAGCACTTGA (SEQ ID NO: 4)

[0196] reverse primer: CCACAGCAAGTCACACATTGG (SEQ ID NO: 5) and

[0197] the PCR probe was: FAM-CGCCTACAAGAAGTTCCCCATGAGGG-TAMRA (SEQ ID NO: 6) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

[0198] For human GAPDH the PCR primers were:

[0199] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7)

[0200] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and

[0201] the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCCX-TAMRA 3′ (SEQ ID NO: 9) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

[0202] Probes and primers to mouse Protein Phosphatase 2 catalytic subunit alpha were designed to hybridize to a mouse Protein Phosphatase 2 catalytic subunit alpha sequence, using published sequence information (GenBank accession number AF076192, incorporated herein as SEQ ID NO:10). For mouse Protein Phosphatase 2 catalytic subunit alpha the PCR primers were:

[0203] forward primer: TCAACAGCCGTGACCACTTTAG (SEQ ID NO:11)

[0204] reverse primer: CGCTATGCCAGAAACTGGATTC (SEQ ID NO: 12) and

[0205] the PCR probe was: FAM-CCAGTTCATTGCATGCTGACGCGA-TAMRA (SEQ ID NO: 13) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

[0206] For mouse GAPDH the PCR primers were:

[0207] forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 14)

[0208] reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 15) and

[0209] the PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATCX-TAMRA 3′ (SEQ ID NO: 16) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Example 14 Northern Blot Analysis of Protein Phosphatase 2 Catalytic Subunit Alpha mRNA Levels

[0210] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0211] To detect human Protein Phosphatase 2 catalytic subunit alpha, a human Protein Phosphatase 2 catalytic subunit alpha specific probe was prepared by PCR using the forward primer CACTGGATCATATCAGAGCACTTGA (SEQ ID NO: 4) and the reverse primer CCACAGCAAGTCACACATTGG (SEQ ID NO: 5). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0212] To detect mouse Protein Phosphatase 2 catalytic subunit alpha, a mouse Protein Phosphatase 2 catalytic subunit alpha specific probe was prepared by PCR using the forward primer TCAACAGCCGTGACCACTTTAG (SEQ ID NO:1l) and the reverse primer CGCTATGCCAGAAACTGGATTC (SEQ ID NO: 12). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0213] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15 Antisense Inhibition of Human Protein Phosphatase 2 Catalytic Subunit Alpha Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap

[0214] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human Protein Phosphatase 2 catalytic subunit alpha RNA, using published sequences (GenBank accession number M60483, incorporated herein as SEQ ID NO: 3, GenBank accession number NM_(—)002715, incorporated herein as SEQ ID NO: 17, and residues 10001-50000 from the complement of GenBank accession number AC007199.1, incorporated herein as SEQ ID NO: 18). The oligonucleotides are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosporothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human Protein Phospatase 2 catalytic subunit alpha mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human Protein Phosphatase 2 catalytic subunit alpha mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB NO 110160 Coding 3 1451 accaaggcagtgagaggaag 58 19 110169 3′UTR 3 2627 caccagtcttgcccattgat 57 20 118885 5′UTR 17 10 ctcgctgaggctccagagct 58 21 118886 5′UTR 17 55 ctctcaccgcagtactcggc 70 22 118887 5′UTR 17 100 ctcgtgtacttctggcggct 64 23 118888 5′UTR 17 124 acacgcacacgccgccgccg 79 24 118889 5′UTR 17 152 tcccgcgccgccgcccgcac 68 25 118890 Coding 17 279 ctcttgacctgggactcgga 80 26 118891 Coding 17 305 ggatttctttagccttctcg 51 27 118892 Coding 17 315 tcttttgtcaggatttcttt 64 28 118893 Coding 17 489 agcagtgtaactgtttcaac 53 29 118894 Coding 17 499 aagagctacaagcagtgtaa 52 30 118895 Coding 17 509 aacgaaccttaagagctaca 62 31 118896 Coding 17 564 tgtgtgatctgtctgctctc 72 32 118897 Coding 17 621 ttccaaacatttgcatttcc 78 33 118898 Coding 17 676 ctgcccatccaccaaggcag 40 34 118899 Coding 17 686 gacagaagatctgcccatcc 49 35 118900 Coding 17 782 agtcacacattggaccctca 82 36 118901 Coding 17 792 gaccacagcaagtcacacat 71 37 118902 Coding 17 877 attaaatgtctcagaaatat 25 38 118903 Coding 17 895 cgtgaggccattggcatgat 57 39 118904 Coding 17 939 cagttatatccctccatcac 44 40 118905 Coding 17 984 tagtttggagcactgaaaat 67 41 118906 Coding 17 1012 tgcagcttggttaccacaac 68 42 118907 Coding 17 1022 gttccatgattgcagcttgg 72 43 118908 Coding 17 1080 tcgcctctacgaggtgctgg 57 44 118909 Stop 17 1122 cattacaggaagtagtctgg 57 45 Codon 118910 3′UTR 17 1152 tcatggcaatactgtacaag 63 46 118911 3′UTR 17 1159 atatggttcatggcaatact 58 47 118912 3′UTR 17 1207 ctgacactttggagttactg 48 48 118913 3′UTR 17 1214 ctattttctgacactttgga 66 49 118914 3′UTR 17 1261 atggcacatcttttggtcca 74 50 118915 3′UTR 17 1262 tatggcacatcttttggtcc 60 51 118916 3′UTR 17 1284 gacaagaggctttgtatttt 70 52 118917 3′UTR 17 1294 ggctgttgatgacaagaggc 67 53 118918 3′UTR 17 1304 aagtggtcacggctgttgat 66 54 118919 3′UTR 17 1309 ttctaaagtggtcacggctg 68 55 118920 3′UTR 17 1314 gttcattctaaagtggtcac 74 56 118921 3′UTR 17 1324 caatgaactggttcattcta 65 57 118922 3′UTR 17 1353 ttcttgaccaacaatgtcgc 68 58 118923 3′UTR 17 1364 cagaaactggtttcttgacc 67 59 118924 3′UTR 17 1374 agcgctatgccagaaactgg 56 60 118925 3′UTR 17 1384 aactacaaatagcgctatgc 54 61 118926 3′UTR 17 1394 aagcaaaagtaactacaaat 25 62 118927 3′UTR 17 1419 cttattatctgcagtctctc 78 63 118928 3′UTR 17 1431 taatgtttacatcttattat 28 64 118929 3′UTR 17 1492 tctacagtcatgctgagtaa 86 65 118930 3′UTR 17 1522 agctccaatgattgtttgct 78 66 118931 3′UTR 17 1529 ttcattaagctccaatgatt 58 67 118932 3′UTR 17 1532 atgttcattaagctccaatg 66 68 118933 3′UTR 17 1710 tcaaaacaactcaccaggtt 65 69 118934 3′UTR 17 1720 acagttctgttcaaaacaac 46 70 118935 3′UTR 17 1830 ccattgatacaattaaaatt  9 71 118936 3′UTR 17 1998 aattgtaatatgtgaaatac 12 72 118937 3′UTR 17 2017 gcacaccaacaatgtgacaa 37 73 118938 3′UTR 17 2029 aacccacaaagtgcacacca 54 74 118939 3′UTR 17 2033 gaagaacccacaaagtgcac 57 75 118940 3′UTR 17 2121 agacttaaatctcaagttat 42 76 118941 Intron 18 2946 aagtgacgtgctgcaaagtt 48 77 118942 Intron 18 5345 agtctttgggttgcatctgt 56 78 118943 Intron 18 5878 atgaataggaagctttcaag 48 79 118944 Intron 18 6840 acccagtctcttagttttcc 58 80 118945 Intron 18 8584 tccaggcgtgagccactgtg 66 81 118946 Intron 18 8824 gctgtcgcttaggctggagt 42 82 118947 Intron 18 10523 gggcgaagtggctcacgcct 36 83 118948 Intron 18 11582 agctgagagcagcaagtggc 54 84 118949 Intron 18 12591 agcttctttttatatcagca 60 85 118950 Intron 18 14058 acacaccaaaaccccatctc 32 86 118951 Intron 18 14626 ataaaggctaatagaggtga 40 87 118952 Intron 18 14682 acattcgcttaaaagccaaa 39 88 118953 Intron 18 18442 ttgacattatcaaattgtcc 55 89 118954 Intron 18 20970 gtgcagtagtatgatcatag 41 90 118955 Intron 18 22349 aatatgctcactttgtcctg 33 91 118956 Intron 18 24971 cagtggtttgttccttttca 60 92 118957 Intron 18 26237 tggcccctctggtgtttctg 32 93 118958 Intron 18 27397 acatgttaatggattcattg 31 94 118959 Intron 18 28742 cgaactcctgatctcaggtg 22 95 118960 Intron 18 28994 aaataaaagttggaatctga  0 96

[0215] As shown in Table 1, SEQ ID NOs 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 65, 66, 67, 68, 69, 70, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 87, 89, 90 and 92 demonstrated at least 40% inhibition of human Protein Phosphatase 2 catalytic subunit alpha expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention.

Example 16 Antisense Inhibition of Mouse Protein Phosphatase 2 Catalytic Subunit Alpha Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap.

[0216] In accordance with the present invention, a second series of oligonucleotides were designed to target different regions of the mouse Protein Phosphatase 2 catalytic subunit alpha RNA, using published sequences (GenBank accession number AP076192, incorporated herein as SEQ ID NO: 10). The oligonucleotides are shown in Table 2. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on mouse Protein Phospatase 2 catalytic subunit alpha mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, “N.D.” indicates “no data”. TABLE 2 Inhibition of mouse Protein Phosphatase 2 catalytic subunit alpha mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB NO 110160 Coding 10 652 accaaggcagtgagaggaag 68 19 118888 5′UTR 10 111 acacgcacacgccgccgccg 75 24 118889 5′UTR 10 138 tcccgcgccgccgcccgcac 85 25 118890 Coding 10 265 ctcttgacctgggactcgga 70 26 118891 Coding 10 291 ggatttctttagccttctcg 40 27 118892 Coding 10 301 tcttttgtcaggatttcttt 76 28 118893 Coding 10 475 agcagtgtaactgtttcaac 44 29 118894 Coding 10 485 aagagctacaagcagtgtaa 58 30 118895 Coding 10 495 aacgaaccttaagagctaca 54 31 118896 Coding 10 550 tgtgtgatctgtctgctctc 70 32 118897 Coding 10 607 ttccaaacatttgcatttcc 94 33 118898 Coding 10 662 ctgcccatccaccaaggcag 64 34 118899 Coding 10 672 gacagaagatctgcccatcc 54 35 118900 Coding 10 768 agtcacacattggaccctca 80 36 118901 Coding 10 778 gaccacagcaagtcacacat 69 37 118902 Coding 10 863 attaaatgtctcagaaatat 13 38 118903 Coding 10 881 cgtgaggccattggcatgat 53 39 118904 Coding 10 925 cagttatatccctccatcac 51 40 118905 Coding 10 970 tagtttggagcactgaaaat 74 41 118906 Coding 10 998 tgcagcttggttaccacaac 78 42 118907 Coding 10 1008 gttccatgattgcagcttgg 88 43 118908 Coding 10 1066 tcgcctctacgaggtgctgg 81 44 118909 Stop 10 1108 cattacaggaagtagtctgg 59 45 Codon 118910 3′UTR 10 1138 tcatggcaatactgtacaag 65 46 118912 3′UTR 10 1193 ctgacactttggagttactg 56 48 118913 3′UTR 10 1200 ctattttctgacactttgga 46 49 118915 3′UTR 10 1247 tatggcacatcttttggtcc 61 51 118916 3′UTR 10 1268 gacaagaggctttgtatttt 71 52 118917 3′UTR 10 1278 ggctgttgatgacaagaggc 58 53 118918 3′UTR 10 1288 aagtggtcacggctgttgat 82 54 118919 3′UTR 10 1293 ttctaaagtggtcacggctg 88 55 118920 3′UTR 10 1298 gttcattctaaagtggtcac 92 56 118921 3′UTR 10 1308 caatgaactggttcattcta 55 57 118922 3′UTR 10 1337 ttcttgaccaacaatgtcgc 93 58 118924 3′UTR 10 1358 agcgctatgccagaaactgg 84 60 118925 3′UTR 10 1368 aactacaaatagcgctatgc 70 61 118926 3′UTR 10 1378 aagcaaaagtaactacaaat 18 62 118929 3′UTR 10 1469 tctacagtcatgctgagtaa 72 65 118930 3′UTR 10 1496 agctccaatgattgtttgct 69 66 118931 3′UTR 10 1503 ttcattaagctccaatgatt 66 67 118932 3′UTR 10 1506 atgttcattaagctccaatg 78 68 118933 3′UTR 10 1664 tcaaaacaactcaccaggtt 59 69 118934 3′UTR 10 1674 acagttctgttcaaaacaac 58 70

[0217] As shown in Table 2, SEQ ID NOs 19, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 65, 66, 67, 68, 69 and 70 demonstrated at least 40% inhibition of mouse Protein Phosphatase 2 catalytic subunit alpha expression in this experiment and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention.

Example 17 Western Blot Analysis of Protein Phosphatase 2 Catalytic Subunit Alpha Protein Levels

[0218] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to Protein Phosphatase 2 catalytic subunit alpha is used, with a radiolabelled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 96 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2 atgcattctg cccccaagga 20 3 2966 DNA Homo sapiens CDS (995)...(1924) 3 aaccaccggc gaggagcggg gcgcgtggaa gcgagccgcg gtccgaggcc caaagaaaag 60 cccaagcctc gcccccgcca tcgcgcccga cgagacacct aggtccgggg acgggtgtgt 120 gccgcggaag tcaggtgcac tgcgcagcac tcccccggta ggtacacgct cctccaccta 180 cgagtgacct aattacaagg tgccagccgc gcccagaggt gggggtggtt aatccaagcg 240 gccactcgct gcccgttcct gcccccaaag atgacggaaa cccacacgat tacagagccg 300 cagcacccca gatgagccac ggggtcgcaa ttctcgtttc cgtgatcgga ctgccaggcc 360 ccaggtgagg agctgagttc atcaccagag cggccttccc aggggaacca gttacaggct 420 gccagtggcc ccggcttcca tccggtctgc gcctgcgcgc ggcccaagcc ctcgcctctc 480 ctggaatagt gctcagggat tagtccggtt cgccgctgtg ccactgcgca tgctccagct 540 ccatccttcc cttcccccac caccccgccc tccgggagcc acgcccaaaa agtcaaggcg 600 cttcagttac cagccggcta cgtggcctgc gctttgaccc ccagtttgcg ccccaactcc 660 ggtcgtgcgg ccgcccgggg agggctctgc agttgcgcag cttgctcccc ggcccttttc 720 ccctccgctc cccgccgcct cctgacgccg ggcgtgacgt caccacgccc ggcggccgcc 780 attacagaga gccgagctct ggagcctcag cgagcggagg aggaggcgca gggccgacgg 840 ccgagtactg cggtgagagc cagcgggcca gcgccagcct caacagccgc cagaagtaca 900 cgaggaaccg gcggcggcgt gtgcgtgtag gcccgtgtgc gggcggcggc gcgggaggag 960 cgcggagcgg cagccggctg gggcgggtgg catc atg gac gag aag gtg ttc acc 1015 Met Asp Glu Lys Val Phe Thr 1 5 aag gag ctg gac cag tgg atc gag cag ctg aac gag tgc aag cag ctg 1063 Lys Glu Leu Asp Gln Trp Ile Glu Gln Leu Asn Glu Cys Lys Gln Leu 10 15 20 tcc gag tcc cag gtc aag agc ctc tgc gag aag gct aaa gaa atc ctg 1111 Ser Glu Ser Gln Val Lys Ser Leu Cys Glu Lys Ala Lys Glu Ile Leu 25 30 35 aca aaa gaa tcc aac gtg caa gag gtt cga tgt cca gtt act gtc tgt 1159 Thr Lys Glu Ser Asn Val Gln Glu Val Arg Cys Pro Val Thr Val Cys 40 45 50 55 gga gat gtg cat ggg caa ttt cat gat ctc atg gaa ctg ttt aga att 1207 Gly Asp Val His Gly Gln Phe His Asp Leu Met Glu Leu Phe Arg Ile 60 65 70 ggt ggc aaa tca cca gat aca aat tac ttg ttt atg gga gat tat gtt 1255 Gly Gly Lys Ser Pro Asp Thr Asn Tyr Leu Phe Met Gly Asp Tyr Val 75 80 85 gac aga gga tat tat tca gtt gaa aca gtt aca ctg ctt gta gct ctt 1303 Asp Arg Gly Tyr Tyr Ser Val Glu Thr Val Thr Leu Leu Val Ala Leu 90 95 100 aag gtt cgt tac cgt gaa cgc atc acc att ctt cga ggg aat cat gag 1351 Lys Val Arg Tyr Arg Glu Arg Ile Thr Ile Leu Arg Gly Asn His Glu 105 110 115 agc aga cag atc aca caa gtt tat ggt ttc tat gat gaa tgt tta aga 1399 Ser Arg Gln Ile Thr Gln Val Tyr Gly Phe Tyr Asp Glu Cys Leu Arg 120 125 130 135 aaa tat gga aat gca aat gtt tgg aaa tat ttt aca gat ctt ttt gac 1447 Lys Tyr Gly Asn Ala Asn Val Trp Lys Tyr Phe Thr Asp Leu Phe Asp 140 145 150 tat ctt cct ctc act gcc ttg gtg gat ggg cag atc ttc tgt cta cat 1495 Tyr Leu Pro Leu Thr Ala Leu Val Asp Gly Gln Ile Phe Cys Leu His 155 160 165 ggt ggt ctc tcg cca tct ata gat aca ctg gat cat atc aga gca ctt 1543 Gly Gly Leu Ser Pro Ser Ile Asp Thr Leu Asp His Ile Arg Ala Leu 170 175 180 gat cgc cta caa gaa gtt ccc cat gag ggt cca atg tgt gac ttg ctg 1591 Asp Arg Leu Gln Glu Val Pro His Glu Gly Pro Met Cys Asp Leu Leu 185 190 195 tgg tca gat cca gat gac cgt ggt ggt tgg ggt ata tct cct cga gga 1639 Trp Ser Asp Pro Asp Asp Arg Gly Gly Trp Gly Ile Ser Pro Arg Gly 200 205 210 215 gct ggt tac acc ttt ggg caa gat att tct gag aca ttt aat cat gcc 1687 Ala Gly Tyr Thr Phe Gly Gln Asp Ile Ser Glu Thr Phe Asn His Ala 220 225 230 aat ggc ctc acg ttg gtg tct aga gct cac cag cta gtg atg gag gga 1735 Asn Gly Leu Thr Leu Val Ser Arg Ala His Gln Leu Val Met Glu Gly 235 240 245 tat aac tgg tgc cat gac cgg aat gta gta acg att ttc agt gct cca 1783 Tyr Asn Trp Cys His Asp Arg Asn Val Val Thr Ile Phe Ser Ala Pro 250 255 260 aac tat tgt tat cgt tgt ggt aac caa gct gca atc atg gaa ctt gac 1831 Asn Tyr Cys Tyr Arg Cys Gly Asn Gln Ala Ala Ile Met Glu Leu Asp 265 270 275 gat act cta aaa tac tct ttc ttg cag ttt gac cca gca cct cgt aga 1879 Asp Thr Leu Lys Tyr Ser Phe Leu Gln Phe Asp Pro Ala Pro Arg Arg 280 285 290 295 ggc gag cca cat gtt act cgt cgt acc cca gac tac ttc ctg taa 1924 Gly Glu Pro His Val Thr Arg Arg Thr Pro Asp Tyr Phe Leu 300 305 310 tgaaatttta aacttgtaca gtattgccat gaaccatata tcgacctaat ggaaatggga 1984 agagcaacag taactccaaa gtgtcagaaa atagttaaca ttcaaaaaac ttgttttcac 2044 atggaccaaa agatgtgcca tataaaaata caaagcctct tgtcatcaac agccgtgacc 2104 actttagaat gaaccagttc attgcatgct gaagcgacat tgttggtcaa gaaaccagtt 2164 tctggcatag cgctatttgt agttactttt gctttctctg agagactgca gataataaga 2224 tgtaaacatt aacacctcgt gaatacaatt taacttccat ttagctatag ctttactcag 2284 catgactgta gataaggata gcagcaaaca atcattggag cttaatgaac atttttaaaa 2344 ataattacca aggcctccct tctacttgtg agttttgaaa ttgttctttt tattttcagg 2404 gataccgttt aatttaatta tatgatttgt ctgcactcag tttattccct actcaaatct 2464 cagccccatg ttgttctttg ttattgtcag aacctggtga gttgttttga acagaactgt 2524 tttttcccct tcctgtaaga cgatgtgact gcacaagagc actgcagtgt ttttcataat 2584 aaacttgtga actaagaact gagaaggtca aattttaatt gtatcaatgg gcaagactgg 2644 tgctgtttat taaaaaagtt aaatcaattg agtaaatttt agaatttgta gacttgtagg 2704 taaaataaaa atcaagggca ctacataacc tctctggtaa ctccttgaca ttcttcagat 2764 taacttcagg atttatttgt atttcacata ttacaatttg tcacattgtt ggtgtgcact 2824 ttgtgggttc ttcctgcata ttaacttgtt tgtaagaaag gaaatctgtg ctgcttcagt 2884 aagacttaat tgtaaaacca tataacttga gatttaagtc tttgggttgt gttttaataa 2944 aacagcatgt tttcaggtag ag 2966 4 25 DNA Artificial Sequence PCR Primer 4 cactggatca tatcagagca cttga 25 5 21 DNA Artificial Sequence PCR Primer 5 ccacagcaag tcacacattg g 21 6 26 DNA Artificial Sequence PCR Probe 6 cgcctacaag aagttcccca tgaggg 26 7 19 DNA Artificial Sequence PCR Primer 7 gaaggtgaag gtcggagtc 19 8 20 DNA Artificial Sequence PCR Primer 8 gaagatggtg atgggatttc 20 9 20 DNA Artificial Sequence PCR Probe 9 caagcttccc gttctcagcc 20 10 1781 DNA Mus musculus CDS (196)...(1125) 10 cgagcggagg aggaggcaca gcggccggcg gccgagcact gcggagcgag ccagcgggcc 60 ggcgccagcg cccagcagcc gcctggggcc gcagaaagca ccccgggaga cggcggcggc 120 gtgtgcgtgt ggcccgggtg cgggcggcgg cgcgggaaca tcgcggaacg gcagccggtt 180 cgggcgggcg gcatc atg gac gag aag ttg ttc acc aag gag ctg gac cag 231 Met Asp Glu Lys Leu Phe Thr Lys Glu Leu Asp Gln 1 5 10 tgg atc gag cag ctg aac gag tgc aag cag ctc tcc gag tcc cag gtc 279 Trp Ile Glu Gln Leu Asn Glu Cys Lys Gln Leu Ser Glu Ser Gln Val 15 20 25 aag agc ccc tgc gag aag gct aaa gaa atc ctg aca aaa gaa tcc aac 327 Lys Ser Pro Cys Glu Lys Ala Lys Glu Ile Leu Thr Lys Glu Ser Asn 30 35 40 gtt caa gag gtt cga tgt cca gtc act gtg tgt gga gat gta cat ggg 375 Val Gln Glu Val Arg Cys Pro Val Thr Val Cys Gly Asp Val His Gly 45 50 55 60 caa ttt cat gat ctc atg gaa ctc ttt aga att ggt gtt aaa tca cca 423 Gln Phe His Asp Leu Met Glu Leu Phe Arg Ile Gly Val Lys Ser Pro 65 70 75 gat aca aat tac ctg ttt atg gga gac tat gtg gac aga gga tat tac 471 Asp Thr Asn Tyr Leu Phe Met Gly Asp Tyr Val Asp Arg Gly Tyr Tyr 80 85 90 tct gtt gaa aca gtt aca ctg ctt gta gct ctt aag gtt cgt tac cga 519 Ser Val Glu Thr Val Thr Leu Leu Val Ala Leu Lys Val Arg Tyr Arg 95 100 105 gag cgc atc acc ata ctc cga ggg aat cac gag agc aga cag atc aca 567 Glu Arg Ile Thr Ile Leu Arg Gly Asn His Glu Ser Arg Gln Ile Thr 110 115 120 cag gtt tat ggg ttc tac gac gag tgt tta agg aaa tac gga aat gca 615 Gln Val Tyr Gly Phe Tyr Asp Glu Cys Leu Arg Lys Tyr Gly Asn Ala 125 130 135 140 aat gtt tgg aaa tac ttc aca gac ctt ttt gac tat ctt cct ctc act 663 Asn Val Trp Lys Tyr Phe Thr Asp Leu Phe Asp Tyr Leu Pro Leu Thr 145 150 155 gcc ttg gtg gat ggg cag atc ttc tgt cta cac ggt ggt ctg tca cca 711 Ala Leu Val Asp Gly Gln Ile Phe Cys Leu His Gly Gly Leu Ser Pro 160 165 170 tcc ata gac aca ctg gat cac atc cga gca ctc gat cgc cta cag gaa 759 Ser Ile Asp Thr Leu Asp His Ile Arg Ala Leu Asp Arg Leu Gln Glu 175 180 185 gtt cct cat gag ggt cca atg tgt gac ttg ctg tgg tca gat cca gat 807 Val Pro His Glu Gly Pro Met Cys Asp Leu Leu Trp Ser Asp Pro Asp 190 195 200 gac cgt ggt ggc tgg ggg ata tct cct cgg gga gct ggt tat acc ttt 855 Asp Arg Gly Gly Trp Gly Ile Ser Pro Arg Gly Ala Gly Tyr Thr Phe 205 210 215 220 ggc caa gat att tct gag aca ttt aat cat gcc aat ggc ctc acg ttg 903 Gly Gln Asp Ile Ser Glu Thr Phe Asn His Ala Asn Gly Leu Thr Leu 225 230 235 gtg tcc aga gct cac cag ctg gtg atg gag gga tat aac tgg tgc cat 951 Val Ser Arg Ala His Gln Leu Val Met Glu Gly Tyr Asn Trp Cys His 240 245 250 gac cgg aac gta gta aca att ttc agt gct cca aac tat tgc tat cgt 999 Asp Arg Asn Val Val Thr Ile Phe Ser Ala Pro Asn Tyr Cys Tyr Arg 255 260 265 tgt ggt aac caa gct gca atc atg gaa ctt gac gac act ctt aag tat 1047 Cys Gly Asn Gln Ala Ala Ile Met Glu Leu Asp Asp Thr Leu Lys Tyr 270 275 280 tct ttc ttg cag ttt gac cca gca cct cgt aga ggc gag cca cat gtc 1095 Ser Phe Leu Gln Phe Asp Pro Ala Pro Arg Arg Gly Glu Pro His Val 285 290 295 300 act cgt cgt acc cca gac tac ttc ctg taa tgaaaatgta aacttgtaca 1145 Thr Arg Arg Thr Pro Asp Tyr Phe Leu 305 310 gtattgccat gaaccgtata ttgacctaat ggaaatggga agagcaacag taactccaaa 1205 gtgtcagaaa atagttaaca ttcaaaaact tgttttcaca cggaccaaaa gatgtgccat 1265 ataaaataca aagcctcttg tcatcaacag ccgtgaccac tttagaatga accagttcat 1325 tgcatgctga cgcgacattg ttggtcaaga atccagtttc tggcatagcg ctatttgtag 1385 ttacttttgc tttcttgaga gactgcagat ataggattaa acattaacac ccgtgagtcc 1445 agttgacttc acttagctgt agcttactca gcatgactgt agatgaggat agcaaacaat 1505 cattggagct taatgaacat ttttaaataa gtaccaaggc ctcccctctt gttgtgtttc 1565 tttcagggat accattaatt taattgtatg atttgtctgc actcagtttc tccccttctc 1625 aaatctcagc cccgcgttgt tctttgttac tgtcagaaaa cctggtgagt tgttttgaac 1685 agaactgttt ccctcctgta agatgatgtt actgcacaag tcaccgcagt gttttcataa 1745 taaacttgag aactgagaaa aaaaaaaaaa aaaaaa 1781 11 22 DNA Artificial Sequence PCR Primer 11 tcaacagccg tgaccacttt ag 22 12 22 DNA Artificial Sequence PCR Primer 12 cgctatgcca gaaactggat tc 22 13 24 DNA Artificial Sequence PCR Probe 13 ccagttcatt gcatgctgac gcga 24 14 20 DNA Artificial Sequence PCR Primer 14 ggcaaattca acggcacagt 20 15 20 DNA Artificial Sequence PCR Primer 15 gggtctcgct cctggaagat 20 16 27 DNA Artificial Sequence PCR Probe 16 aaggccgaga atgggaagct tgtcatc 27 17 2181 DNA Homo sapiens CDS (210)...(1139) 17 agagagccga gctctggagc ctcagcgagc ggaggaggag gcgcagggcc gacggccgag 60 tactgcggtg agagccagcg ggccagcgcc agcctcaaca gccgccagaa gtacacgagg 120 aaccggcggc ggcgtgtgcg tgtaggcccg tgtgcgggcg gcggcgcggg aggagcgcgg 180 agcggcagcc ggctggggcg ggtggcatc atg gac gag aag gtg ttc acc aag 233 Met Asp Glu Lys Val Phe Thr Lys 1 5 gag ctg gac cag tgg atc gag cag ctg aac gag tgc aag cag ctg tcc 281 Glu Leu Asp Gln Trp Ile Glu Gln Leu Asn Glu Cys Lys Gln Leu Ser 10 15 20 gag tcc cag gtc aag agc ctc tgc gag aag gct aaa gaa atc ctg aca 329 Glu Ser Gln Val Lys Ser Leu Cys Glu Lys Ala Lys Glu Ile Leu Thr 25 30 35 40 aaa gaa tcc aac gtg caa gag gtt cga tgt cca gtt act gtc tgt gga 377 Lys Glu Ser Asn Val Gln Glu Val Arg Cys Pro Val Thr Val Cys Gly 45 50 55 gat gtg cat ggg caa ttt cat gat ctc atg gaa ctg ttt aga att ggt 425 Asp Val His Gly Gln Phe His Asp Leu Met Glu Leu Phe Arg Ile Gly 60 65 70 ggc aaa tca cca gat aca aat tac ttg ttt atg gga gat tat gtt gac 473 Gly Lys Ser Pro Asp Thr Asn Tyr Leu Phe Met Gly Asp Tyr Val Asp 75 80 85 aga gga tat tat tca gtt gaa aca gtt aca ctg ctt gta gct ctt aag 521 Arg Gly Tyr Tyr Ser Val Glu Thr Val Thr Leu Leu Val Ala Leu Lys 90 95 100 gtt cgt tac cgt gaa cgc atc acc att ctt cga ggg aat cat gag agc 569 Val Arg Tyr Arg Glu Arg Ile Thr Ile Leu Arg Gly Asn His Glu Ser 105 110 115 120 aga cag atc aca caa gtt tat ggt ttc tat gat gaa tgt tta aga aaa 617 Arg Gln Ile Thr Gln Val Tyr Gly Phe Tyr Asp Glu Cys Leu Arg Lys 125 130 135 tat gga aat gca aat gtt tgg aaa tat ttt aca gat ctt ttt gac tat 665 Tyr Gly Asn Ala Asn Val Trp Lys Tyr Phe Thr Asp Leu Phe Asp Tyr 140 145 150 ctt cct ctc act gcc ttg gtg gat ggg cag atc ttc tgt cta cat ggt 713 Leu Pro Leu Thr Ala Leu Val Asp Gly Gln Ile Phe Cys Leu His Gly 155 160 165 ggt ctc tcg cca tct ata gat aca ctg gat cat atc aga gca ctt gat 761 Gly Leu Ser Pro Ser Ile Asp Thr Leu Asp His Ile Arg Ala Leu Asp 170 175 180 cgc cta caa gaa gtt ccc cat gag ggt cca atg tgt gac ttg ctg tgg 809 Arg Leu Gln Glu Val Pro His Glu Gly Pro Met Cys Asp Leu Leu Trp 185 190 195 200 tca gat cca gat gac cgt ggt ggt tgg ggt ata tct cct cga gga gct 857 Ser Asp Pro Asp Asp Arg Gly Gly Trp Gly Ile Ser Pro Arg Gly Ala 205 210 215 ggt tac acc ttt ggg caa gat att tct gag aca ttt aat cat gcc aat 905 Gly Tyr Thr Phe Gly Gln Asp Ile Ser Glu Thr Phe Asn His Ala Asn 220 225 230 ggc ctc acg ttg gtg tct aga gct cac cag cta gtg atg gag gga tat 953 Gly Leu Thr Leu Val Ser Arg Ala His Gln Leu Val Met Glu Gly Tyr 235 240 245 aac tgg tgc cat gac cgg aat gta gta acg att ttc agt gct cca aac 1 001 Asn Trp Cys His Asp Arg Asn Val Val Thr Ile Phe Ser Ala Pro Asn 250 255 260 tat tgt tat cgt tgt ggt aac caa gct gca atc atg gaa ctt gac gat 1 049 Tyr Cys Tyr Arg Cys Gly Asn Gln Ala Ala Ile Met Glu Leu Asp Asp 265 270 275 280 act cta aaa tac tct ttc ttg cag ttt gac cca gca cct cgt aga ggc 1 097 Thr Leu Lys Tyr Ser Phe Leu Gln Phe Asp Pro Ala Pro Arg Arg Gly 285 290 295 gag cca cat gtt act cgt cgt acc cca gac tac ttc ctg taa tgaaatttta 1 149 Glu Pro His Val Thr Arg Arg Thr Pro Asp Tyr Phe Leu 300 305 310 aacttgtaca gtattgccat gaaccatata tcgacctaat ggaaatggga agagcaacag 1209 taactccaaa gtgtcagaaa atagttaaca ttcaaaaaac ttgttttcac atggaccaaa 1269 agatgtgcca tataaaaata caaagcctct tgtcatcaac agccgtgacc actttagaat 1329 gaaccagttc attgcatgct gaagcgacat tgttggtcaa gaaaccagtt tctggcatag 1389 cgctatttgt agttactttt gctttctctg agagactgca gataataaga tgtaaacatt 1449 aacacctcgt gaatacaatt taacttccat ttagctatag ctttactcag catgactgta 1509 gataaggata gcagcaaaca atcattggag cttaatgaac atttttaaaa ataattacca 1569 aggcctccct tctacttgtg agttttgaaa ttgttctttt tattttcagg gataccgttt 1629 aatttaatta tatgatttgt ctgcactcag tttattccct actcaaatct cagccccatg 1689 ttgttctttg ttattgtcag aacctggtga gttgttttga acagaactgt tttttcccct 1749 tcctgtaaga cgatgtgact gcacaagagc actgcagtgt ttttcataat aaacttgtga 1809 actaagaact gagaaggtca aattttaatt gtatcaatgg gcaagactgg tgctgtttat 1869 taaaaaagtt aaatcaattg agtaaatttt agaatttgta gacttgtagg taaaataaaa 1929 atcaagggca ctacataacc tctctggtaa ctccttgaca ttcttcagat taacttcagg 1989 atttatttgt atttcacata ttacaatttg tcacattgtt ggtgtgcact ttgtgggttc 2049 ttcctgcata ttaacttgtt tgtaagaaag gaaatctgtg ctgcttcagt aagacttaat 2109 tgtaaaacca tataacttga gatttaagtc tttgggttgt gttttaataa aacagcatgt 2169 tttcaggtag ag 2181 18 40000 DNA Homo sapiens 18 ggccttgcca aacacgttgt cttggataaa ggaggatttt cagtcctgct attggctgat 60 ggggcctcca gtgccttctt cagcctgttg gtggggatga tgccctcaga gccgcagagt 120 gagcagtctc ctcccaagca tcgctgagca cttgggtgtg gaagggttgc ctgatactga 180 gggtggggtt cctggaagac aggcgatcag gttcagctct tgccttgccc tttatggtta 240 aatgttcttt ggaaagcttc tatatctttc aaatcctggg ggcattatga tctctcttct 300 gcctatccca aaccatcttc agccagtact gttgggagaa tacaagctcc agggcatggt 360 ccctggtccg tctggatgac ccctccggat ctggcatata gctggcgctc tatggaacca 420 gatatttatg taaaagcagg gacaatggaa tgtgaaaacg tttcgcaaac tccaaagcga 480 ttcataacca gacagaggtc caatcctagg gtatacgagc tctctctttt aagcacgtat 540 cacagctgct tctcacgcca ccgagacgcg gtcaggacag gtgcatccat cttctctggg 600 cttcccctct tgaaagtagg ccagggcgcg caggggttga gggtctctca ttcccagaga 660 gaattaaact tggaggaaag caaccaccgg cgaggagcgg ggcgcgtgga agcgagccgc 720 ggtccgaggc ccaaagaaaa gcccaagcct cgcccccgcc atcgcgcccg acgagacacc 780 taggtccggg gacgggtgtg tgccgcggaa gtcaggtgca ctgcgcagca ctcccccggt 840 aggtacacgc tcctccacct acgagtgacc taattacaag gtgccagccg cgcccagagg 900 tgggggtggt taatccaagc ggccactcgc tgcccgttcc tgcccccaaa gatgacggaa 960 acccacacga ttacagagcc gcagcacccc agatgagcca cggggtcgca attctcgttt 1020 ccgtgatcgg actgccaggc cccaggtgag gagctgagtt catcaccaga gcggccttcc 1080 caggggaacc agttacaggc tgccagtggc cccggcttcc atccggtctg cgcctgcgcg 1140 cggcccaagc cctcgcctct cctggaatag tgctcaggga ttagtccggt tcgccgctgt 1200 gcgcactgcg catgctccag ctccatcctt cccttccccc accaccccgc cctccgggag 1260 ccacgcccaa aaagtcaagg cgcttcagtt accagccggc tacgtcgcgc ctgcgctttg 1320 acccccagtt tgcgccccaa ctccggtcgt gcggccgccc ggggagggct ctgcagttgc 1380 gcagcttgct ccccggccct tttcccctcc gctccccgcc gcctcctgac gccgggcgtg 1440 acgtcaccac gcccggcggg cgccattaca gagagccgag ctctggagcc tcagcgagcg 1500 gaggaggagg cgcagcggcc gacggccgag tactgcggtg agagccagcg ggccagcgcc 1560 agcctcaaca gccgccagaa gtacacgagg aaccggcggc ggcgtgtgcg tgtaggcccg 1620 tgtgcgggcg gcggcgcggg aggagcgcgg agcggcagcc ggctggggcg ggtggcatca 1680 tggacgagaa ggtgttcacc aaggagctgg accagtggat cgagcagctg aacgagtgca 1740 agcagctgtc cgagtcccag gtcaagagcc tctgcgagaa ggtgagctgt agtacggctg 1800 cggacagccg ccgcggggcc gagcccgccg aggaaaaggc ggccgtgagg agggtgcaac 1860 atggcggagg ccgccggtcc gggccccccg agtctccgag accggcgccc atcctggccg 1920 gcggcggctt cctaacgacc cggcgccccc tgcctcccgc gcagggattg gttgccgccg 1980 ccgcctaaaa tggcgccgtt cactcgactc ctgggctttt gagtcagccg gccttggcgc 2040 cagacgaggt ggcaggggga gcggagaccc tgtgggtcat cgtcagggac ggttttgagg 2100 gtgagctggc ttggattctg cggcttggga atggagcctg ggcctctccg gtgggctgag 2160 taagagtctt tccgctggta cccaggccta gttaggtcca gagctgccca gaggatcccc 2220 caaaggcagt cttgttaggc cgtatcccag aacataccta aaggtcacac agctttagta 2280 ccccttggtg gtttgtatta tactcaccaa gcgatcttct ccagaaatca aggtaagggg 2340 tgtctttaag aatttttaaa ttattctctt aacctaattt ttacgaaggt catggtggta 2400 gctataaggg aggagtgtaa cttatttttc atagttggaa gaaatcggga atttggtgag 2460 tcatactaca taaggagctt ttggattgga aatcaagtgt cggttgaata tgatttatat 2520 gctgactcaa agccttctct aagcttaaca cacttggcat tttgccgttt atttttaaaa 2580 tgaaccacat aaaatggaag agcggtaaat tttgtatctt tgttgcaaaa attacttgat 2640 agtattttgt ggctgcaagt agtagtagtg ataataactg tcgactgaga ttgtattcca 2700 gacattgtgc ttagtgttta aataatgtct cacatacact gaaaatagaa tgttgtagtc 2760 cacttagtaa cactaaactt tatattagct aattcccttt tgttttcccc tgggggttgg 2820 ggaaacaggt agttaattta gctattagct ttgtgttgta tttttaaatt cttgtaactc 2880 agttttctaa agtacacaaa atgctgatct ctgctgtgaa cattttaact tcccttttca 2940 aaattaactt tgcagcacgt cactttagta cagaatagtg gagtatcata ttagcatttt 3000 gtatccgtga aaagaattaa tggagaacta ttatcctgat ttgtttctgg tttgatgtga 3060 aaattaggtt catgagtttg acgtatatgt ttccaagaca ggctttttta gaaaccatgt 3120 tgtgaacaat tggaatttaa gtaagtcaga tttagatttg tttctcccaa gcctgaacaa 3180 aactactact agtggatgag gtggcacatc atctgttgga gatgcctttt aatggtagcg 3240 atgtattgaa tctcctgttt ctttaccctc tcacgtcaaa tgaggttggt atttatttta 3300 aaggtttaaa attggccttt aaaaatgaat gtatattgcc aggtcttaat ttctaggtac 3360 tgtacatgat gatcttaccg atttttggaa ttcagtgcat tatatgaaag acttttaagt 3420 cagtagttgg ccagttgact agtcttttga agaaaacgtg gaaaggaggg aggtgatgac 3480 gtaactggaa gactggactg cgagttagat ttggacatga gtccttctgc tcttccttga 3540 gtaattgtga cagactttgg gacagccact tcacttgtcc ttgttttcct gtctataaac 3600 taaaatgtgt tctgagatta tacagtcaat tccgttttaa attagtagtg tgtttaatat 3660 tgaacatcta ttgaatatct aagattgcat actcttttga gggaaatgaa aagcaatgaa 3720 gaataagtat gcttattgtc gaggagctat gaatctagtt tgaaagaaaa gcatggtatg 3780 aaaaggtgtg ggagatggag catggaagta gagggtttgt gttggaaact gttgtagaag 3840 ataagataat actggatgct agagtggggt gggtgaagct gatgcatcta atattgcttt 3900 ccacatcttt gtaaaactaa gatgtagtga ggtaaatctc cattgcttgc gctgcttacg 3960 caaattttag ttctgagtgc caaaaaagat ggaaataata tttgagagta ggtgttacat 4020 aaaagtttct gtggaatgga tccgttttgg tggatatcat tatacaacct tcatactgtt 4080 tataactggt aagtaagtat ttcaactttc aggatgatat taaacttcca aactaaatta 4140 atttgaagac agttttttgg gtatgtagaa tccatgaact gatgtttttt gtacaagtct 4200 ttctagtaaa acaaaagctg tctttcactg ttaatatttg tgtgccaatg gcatctctgg 4260 gtgaaagcta catatgtgct ttttggtgtg attgccttct aaagagtaat tttgaaaatt 4320 tgagggctaa tttttttcat tagtgtataa taatagtatt ttttgagaaa aaagatactt 4380 gttaatagtt gataatttct tcaatcataa gaaggaatga taatgtaaaa gccttccttc 4440 tacccaccta aatgcctgat ttgaagggga aatatttaaa tagtaaaaat agatgtatac 4500 cataagaggt cattgttact aaaaaatcct gaggccattt ttttgttatc ccatcaacaa 4560 ttaacagtgt tacatgttca gaactgagag aatctaagta tgcatttata aaagacaaga 4620 gtgaacatgg tttttatgtt actgactagc taaaaagaat tttaagatgt gattaagact 4680 tatacaaaag ccaaggttgt tagtgtatag tatactttgt catcttgaga atcataattt 4740 gttgatagtt taataatttg gtgtgcagct ttgcctcagg ctcagttcta taatcagcat 4800 tacatcagta taactggccg ggtatgtttc aatctacaga ttgaaaggag taaatagtat 4860 caacttgctt ttgcattgac tacagtaaac aatcttttat tatgtgactt cagctttgtt 4920 ttagcttggg tatgatttct tggctttgtg cttaccaaac atgtggagaa cgttgtttga 4980 tagccagatt tttttttttc aaagaagttc tttacctagg tatgctgttt tccattgctt 5040 tgcaagcact ataaataaag tgcagtcatt agtcattaaa atgttagttt taacatttgt 5100 tacgtgagta tggaaaaaac aaactgcttc cttctaatta gatgagctga aaaatatttg 5160 accaagatga cattcttgta tgaattgggt aatatgttgc tgtgagtatg tgtgtacgtt 5220 tcattttaaa gggtgtttgg ctcatttctc tagaagtgat ttgaatattg aggacaagca 5280 ggtgggggaa gcatgttaca gaacattatt tttatcatac ttttataaga tattgttctc 5340 agttacagat gcaacccaaa gactcagaaa ggtaaagtga cttgactaaa ggggctaaaa 5400 gtgagattgc caaggcttgt ggattctaag cctaatgttc tggtattgct tcacagatcc 5460 ccttctgttt tcagaggtag tgttagtagt gaaagatgta ggtaaattgg ggccaagcta 5520 aattgggaag tcaacacttt cataaaataa aacttttttt tttttttttt tttgagataa 5580 ggtcttactc ttttgcccag tctggagtgc agtggcgcaa tctcggctca ctgcaacctc 5640 cacctcccag gttcaagcag ttctcctgca tcagtctccc tagtagctgg gattacaggt 5700 acccaccacc atgcccagct gttttgtatt tttagtagag atgggatttc accatgttgg 5760 ccaggctagt ctcgaactcc tgaccttcag tgatccaccc acctcagcct cccaaagtgc 5820 tgggattaca ggcttgagcc agcgcgcctg gccaaaacat tatttttatt ttcattgctt 5880 gaaagcttcc tattcatgtt acatactgtt gttccgcttt gtcttggaaa gagtaattat 5940 tattcagttt tggatgtagc tgacaacatt tgcccctcaa atgagattgg tgagatgaaa 6000 accctagata atttcccttg tattcttagt ataaatcatg atccagagac tgagctctaa 6060 agtattctaa taaagtataa ctgacttttt atattgttta ttgcaactta taaaatgcct 6120 cttttcagca gggcaaggtg gctcacacgc ctgtaatcct agcactttgg aaggccacgg 6180 caggcggatc accaggtcag gagaccaaga ccaatcctgg ctaacacggt gaagccccgt 6240 ctctacttaa aaatacaaaa aattagccgg gcgtggtggc acacacctgt agtctcagct 6300 actggggagg ctgagacagg agaattgctt gaacctggga ggcggaggtt gcagtgaacc 6360 aagattgtgc cactgcactc cagcctgggc gacagaggga gaccccattc cccccggccc 6420 caaaaaagcc tcttttcatc ctcttggctt ccacagtcaa atgtcatgca tgtgtgtatt 6480 tgtttcatgg tctcctaggc aggaagctgt aatgcagtta gtttggagtt gacttcatga 6540 ggaaacagaa aaggtgaaca ttcccactat tcagaaacac aggttcccca accctcccta 6600 cgttctccac tgggaccaga attgtatgtt ttcagaaaat tggctgtaag gctaagttga 6660 agctcatgta ctgtagaaag aatgaaggac ccttcaggag tatgtgggat aacatttgta 6720 ttaccagtct ggtgtggccc tgttttatgg cagcaaatat gttccctatc tcatggtaag 6780 tcaggtttgt cagagttacc tgtgagatag cctttgctta gtttggaagg ggtaagttag 6840 gaaaactaag agactgggtt tggtggggtc ttttatcagg gtactgagga gagaggcaaa 6900 gctatatgta acagggagac actatttatt ctttctgctt tgacccattt ctcttacttc 6960 attatcatcc ttgcctgaga aagctgtcaa cctccactca tacacaggca gatctgaatt 7020 ggatgagaac catgaaggag gaagattttt gtttttgtgg gctttttttt tttttttttt 7080 tttttttttt gagatagagt ctcacagtcg cccaggctgg agtgcagtgg tgtgatctcg 7140 gctcactgca ccctctgcct cccaagttca agcagttctc ctgcctcagc ctcccaaata 7200 gttgggatta taggtgtgtg ccaccacacc cagctaattt ttgtattttt agtagagaca 7260 gggtttcacc atgttggcca ggctggtctt gaactcctta cgtcaagtga tccacccatc 7320 tcagcctccc aaagtgctag gattacaggc atgagccact ggcaccagac ctgtttttgt 7380 tttaaataga ttttcttttg gcttctgggg cagaagtggt agaccaggct gaagaaggga 7440 gaggtgtcta ttgtcagtaa atggctgaga ggtggggttt gaaagaatgg taaagaacaa 7500 cctgagatta actgcttttt tttttttttt ttttgatacg gagcctcgct ctgttgccca 7560 ggccagagta cagttgcatg atctcggctc actgcaacct ttgtctccca cgttcaagtg 7620 tttctcctgc ctcagcctcc tgagtagctg ggattacagg catgtgccac catgcctagc 7680 taattttttt atttttagta gagacagggt ttcatcatgt tggccaggct ggtcttgaac 7740 tgctgacctc aagtgatccg cccgcctcag cctcccaaag tgctgggatt acaggtgtga 7800 gccactgtgc ccagctaact gcttaacttc ttcacattgc agtattattg cctgtttatt 7860 ggtctgttgt ggattgtggt ttagaatgga tgaacaaggc agtgacacag atgcgtgtat 7920 atctgaatct gggtattttt taaactaaat gtgaccagtt ggccagtccc tcttgcaccc 7980 tgtgttgcca cttctgcctt agtttagtca tttagttgct actacagatg gggattttta 8040 ccctgctggt gttgaggctg gaaggttgca aacctttgtc ctagatgggg acgaggagta 8100 gaatgggaga caagcagtag catgtgttta cctggccaaa ggcctgcaaa atgtagttca 8160 cagaggttat ggagtgtcat tgatgctatc gtgtgacagt gtacatttga ttttggaacc 8220 agtatttaat aaaccttcat tgtatgcatt attgtcacta ttgagctttg tctaaaaagc 8280 actaccttat tctcgtgtat aaggatttct tagtttggtt ttgctctaat taatagcaca 8340 gttttggatt agtcaggaga ttgactttat agttaattct agatagggaa tgttttagaa 8400 tgcttgtgcc ggtggtgatc aagtaactta tttttctgcc actaatctgt aaatgagcaa 8460 taatgcccac tatctatttc ttttggctgg ctttacttaa taaaataggc ttaagacatg 8520 ctttaggatg gggtagaatt ctagtaatgg aatcaacctt aaagaccatc taggctggcg 8580 gggcacagtg gctcacgcct ggaatcccag cactttggga ggctgaggtg ggtggatcac 8640 gaggtcagga gatggagccc atcctggcta gcacggtgaa accccgtctc tactaaaaat 8700 acaaaaaatt tagccaggca tggtggtggg cgcctgtagt cccagctgct cgggaggctg 8760 aggcaggaga attgcttgaa cccagcaggc agaggttgca gtgagccgag atcgtgcccc 8820 tgcactccag cctaagcgac agcaagactc catcttaaaa gaaaaaaaaa aaaaaagaaa 8880 gaaacaacca tctagactaa ttcatcttac acagatgagg gaggaaactg agacccagag 8940 tggttgaatc ttgttcatga tgtattattg tgacaatttt ttaataagta gatatatagc 9000 agcatttctc agttgtgtag tagattaaaa cttctttttc tatgcaggaa tgattgggag 9060 tgtttaattc attcacatat gtaggttagt tggtaaaggt tgagtttagt ctattgtgca 9120 tatgagacat agggctttaa ggatctttcc ttttggtcag aggcaaaggt gatattgcag 9180 aaacagatgg gaaaactcac aaattttata ttttctttta ttacactttt agaattatcc 9240 ctcattccca acatttttct tttatttagg tcctaagcct tagagccaaa cagaaaatac 9300 tagttactag aatattacct ggagggttaa acttttaaaa tataaccata ctgattttaa 9360 tcaaagaaga ttgaaatgtt tcgtgaaata ttagtggtat ctgttcagtg tagttaatga 9420 aagacaagta gtcatgcacc acataatgac attttggtca gtggtggact gcatgtgatg 9480 gtggtcttat gaaattataa tggagctgaa aaattcctat cacctggtga ccttgtaatg 9540 tcatagtgta acactttata ttgtttatgt ttagttatac aaatacttaa cattatgtta 9600 acagttgcct acagcattca gtacagtaaa tgctgtacaa gtttgtagcc taggagcaat 9660 aggctatcca atagcttagg tttgtagtag tctatactat ctagatttgt gtaagaccac 9720 tctgtgatgt tcacacaaca actaaataat ttgatgtatc cctgcgatta agcaacacat 9780 gattagtatg aagaatggtt tgatagttga aggaaattaa ctcaaaactc atttgcttcc 9840 agggctagta atatgttcta catcatgtat caccccacgc ctcaaactct agccctgtac 9900 actaacttat cacaaacctt ggattttttt gcacaattat caaaatgaca tatcaaacca 9960 accgtacatc ttgacataaa aacagtatga gacaaaatga aattctcttg ggatattgaa 10020 aaaatttttt ttgcctgtga atttagtaat tttacttatt aagaattttc aaatgatttc 10080 agttcttaaa ctgaaaaata aaattcaaaa attaattgca ttttttccag tgtttcttct 10140 atgtctttat attggtagaa ctgtccaact atatggtaag gatgtgaaat gttaaattta 10200 cttgaactag agttacaatg aacattagaa ttaataatca gacttctttt ttttttcttt 10260 ttttgagaag ggagtttcgc tcttgttgcc caggctggag tgcagtggca cggtcttggc 10320 tcaccgccac ctccaccttc cagattcaag caattctcct gccccagcct cccgggtagc 10380 tgggattaca ggcatgcgcc accacaccca gctaattttt gtattattag agatggggtt 10440 tctctgtgtt ggtcaggctg gtcctcaaac tcccgatctc aggtgatctg cctgccttgg 10500 cctcccaaag tgctgggatt acaggcgtga gccacttcgc ccggccaata atcagacttt 10560 tagtagtaaa attactttca aaaatggaga ttcctttgtc aaatagtact attattagta 10620 ctaagattgt aaagaggtta tttgtgtctg attttgagtt tgatgctaca gagaagacag 10680 gttaggcctg cattgacgtt tttcatcaag ggcagaattg atgatacaga aacttgattt 10740 ctgtctaggc atggtggctc atacctacaa tcctggtgct ttgggaggcg gaggctggaa 10800 gctcactcga gactaggagt ttgagaccag cttgggcaat gtagtgagac caccgtttct 10860 acaaaacaaa aaacaggttt ctttagttaa gtataaatca ttgatctgtg attaatccaa 10920 acactttttc ctcagtatag accttggggt tcttcggtgt ttgactacct tcttttgaag 10980 gaaaccacct tttttttttt tttttttttt tttgaggcag tctcactctg ttgcccaggc 11040 tggaatgcag tggcacaatc tcagcccact gcaacctccg cctcatgggt tcaagtgatt 11100 ctccagcttc agcctcctga gtagctggca ttacaggcat gcaccaccat gcccagctaa 11160 tttttgtatt tttagtaggg gagggggtgt ttcgccatgt tgcccaggct gttctcaaac 11220 tcctgacctc aagcgatcca cccaccctgg cctcccgaag tgctgggatt acaggcatga 11280 tccaccgtgc ccacccgaaa ccacttgtaa tataccttta ggagaatgct tttagttaca 11340 tcatgatccc tttcatgtag ctaacaacaa aaatgaatat taaccccaaa tattggaact 11400 cgtattttga agcaagttat ttcttggtat actttagtgc cagaattggt actgttatgt 11460 gattaactgt ttttgagact aagaaaatat tttgctggac atctgatgaa attatcttaa 11520 aaacagtgct tgggtgtttt ccctggagtg tagtggcaag agtactctgt tgtccatcag 11580 agccacttgc tgctctcagc tggccttgct gttgaatggc ataatctgat ttcgttggag 11640 tacccaagtc ctaagatttg aagaataagg gttattatct cagtctcatc ttttctctga 11700 gatagcattt gttcaggaaa tgttatctgt tttcaatggt gaagggagtg agagtctgct 11760 taggagttac tgtatacatt aaacattggt gaccggcctt taaagaatta gaggctaggc 11820 ctggtgactt acacctataa tcccagcaga ttgggaggcc agggcaggag gatatcttga 11880 gcccgggagt tcaagaccag cctaggcagc atagtgagac cctgtctcta caacaaaatt 11940 tttacttttt tatttttatt tttatttttt gagatggagt tttgctctgt cacccaggct 12000 ggagtgcagt ggcatgatct tggctcactg caacctccac ctcctgggtt caagcgattc 12060 tcctgcctca gcctcctgag tagctgggac cacaggcgag ggctaccatg cccagaatat 12120 ttttgtattt tttttttggt agagatgggg tttcaccact ttggccaggc tggtcttgaa 12180 ctcctgacct caagtgatcc acctgcctcg gcctcccgaa gtgctgggat tacaggcgtg 12240 agccactgca cccagcccta cgaataattt ttaaaaataa attagttggg tgtggtggca 12300 cacctgtggt tctagctact ctgtgaggct gaggcaagag gattgcttgg gtccaggaga 12360 tctagactgc aatgagctgt gatcatgcta cagcactcga agcctgggga acagagcaag 12420 aacttgtctc aaaaaaaaaa aaaaaagtaa ataaatatta gacacttctc aagccctagg 12480 tcccaattga ttgatttttt tttttcttgg atgcaggatt ccaaaatact tcaaggtcat 12540 tattaaacat ttaaagttaa tgtcataatg ttaacgatgc aagtttaaga tgctgatata 12600 aaaagaagct tgagtagttg tcgtgtaatc ttaggattaa agtatagatt ttaaaatgaa 12660 aaactcttag aaaatggcaa gacgtgataa cttttcttaa taattgtagg agaaaaatta 12720 caaagcaaaa atgggcatag aaaatttctg gctatatatt taaaaaataa gagaaaatgc 12780 caaaaagtta cttaaaataa cagtttaaaa tagtttatac aaatacttca gaacatcatc 12840 tagctcttaa tgatcagatg ttcaaaaata ttcatggaca acttgtataa gaggagaaat 12900 aactgatggt aaataaacat ggaataaaaa gtttgtactc agaaatgaaa aatgccaatt 12960 aaattagtga taccacattg tatatctgaa atagctgaga atgagtatat tctttcattt 13020 attattgtat aacaaattgg cataattgag ttttatagta catatgaaac aaaaaaagtt 13080 aatgctattt tgccctgtaa tcccacaatt aaagcttttt gaaaggaaat aattcaaatg 13140 agcaataata ctgtacacgt ataggtctat ttagaagggc attatgtagt tttaaaaaat 13200 cactataatg tagaaacgag ttagtatgaa actttaggac acaaaatgag atgccagtca 13260 attataagta agaggtctgt gtgggtaaaa actgaaaggt aatagaaatg agaagaattc 13320 tggttggtaa gaataagata gttatatatc ttaaagcttt cagtgaaatc ttaacctttt 13380 aatttttttt ttagcctttt tttgttagtt ttgagagaga gtcttactgt gttgcatagg 13440 ctggagtgca gtggcacgat ctcagctcac tgcaacctct gcctcctggg ttccaataat 13500 tctcatgcca cagcctccca agtagctggg attacaggcc tgtgccatct tacctggctg 13560 atttttgtat ttttagtaga gatggggttt tactgtgttg gccaggctgg tcccaaactc 13620 ttgacctaaa gtcatccacc tgctttggct cccaaagtga tagaattaca ggcgtgagcc 13680 actgtgcctg ggcttttcag ccttttttta atgcaaagaa ttgtgacatg gttgtaatca 13740 ttttgtgctt ttatttattt attttgagat ggtgttgttg cccaggctga agtacagtga 13800 catcatcata gttcactgga accttgaact cctgggctca agtgattctt ctgcctcagc 13860 ctccctagca gctgggacca cagagtgcac caccatgcct agctaatttt tatttttcgt 13920 agagacaggg tctcgccaca ttgcccaggc tgatcttgaa ctcctgagct caagcaatcc 13980 tcctgccaca gccttccaag tagctggtac tacaggtgtg taccactatg cccagctaat 14040 attttaattt ttttgtagag atggggtttt ggtgtgttgc ccaggctgag cagtaatatt 14100 ttgaaaggaa tctttttttt tttttctgag cagtaggtct caataatgca cttaaaatat 14160 tcagtatacc gtgctgtaag cagatgtgct gtcatccagg ctttgttgtt ccatttctag 14220 agcacaggcg tagtagattt agcataattc ttaagtgcta aagaatggct taaatgcttt 14280 cagaatggtg aatgagcctt ggcttcaact gaaagtcacc agctacatta gcctctaaca 14340 agagtcagcc tgtccgttga gggtttgagg gcaggcattg acttctctag ctagtaaagt 14400 cctaggtggc atcttcttcc actagaaggc tatttcgtct acattgaaaa cctgttgttt 14460 agtgcaacca ccttcatcac ttatcttagg tagatcgtct gggaaacttg ttgtagtttt 14520 tatattggta cttgatgcct tatcttgccc ttttatgtta tggaaatggg ttcttaaatc 14580 tcatgaacca gtctgctagc tttaagctta acttctgcag cttcctcacc tctattagcc 14640 tttatagaat tgaagaatta ggacctacct ctgaattagg ctttggcttt taagcgaatg 14700 ttgtggctga tttgatctat ccagaccact aaaactttgt ccgtatctgc aataagactg 14760 tttggcttac ttactatttg tgtgttcact gaagtagcat ttctaatttc cttaaagaac 14820 ttttcctttg catttatacc ttagcagttt ggtgcaagaa gcctaccttt aggcctgtct 14880 ggagtttcca tatgccctct cctaagttta ataatttcta acttttcatt taaagtgaga 14940 gatatgcaac tcttcctttc acttgagcat ttggaggcca ttgtagggtt attaattggc 15000 ctaatttcaa tattgtgtct caggggatag ggaggcccaa ggagagggag agacagaatg 15060 gccagtcagc agcagtaaga atatatacga catttattaa gttcattgtc ttatgggcac 15120 ggttggtagt gccccacagc aattacaata gtaacatcaa agatcactga tcacatatca 15180 ccataacaga tacaatagta atgaaaaagt ttgaaataat tttagaagta ccaagatgtg 15240 acaaagacaa gaagtgagca catgctggtg ggaaaatggt gctgataaac ttgctccaca 15300 cagggttgcc atgaagcttt gattgattaa aaaaatgcaa catatgcaaa gtgcaataaa 15360 gagaagcaca gtaaaatgag gcatgcctgt agttattgtg tgctcttaag gaaattacta 15420 ttactatttt tgagatggag tctcactgtc acccaggctg gagtgcagtg gtgcggtctc 15480 cgctcactgc aacctcgcct cccaggttca agtgattctc ctacctcagc ctcccaagta 15540 gctgggatta caggcgcctg ccaccacgcc tggctaattt ttttgtattt ttagtacaga 15600 tggggtttca ccatgttggc caggctggtt tcgaactccc gacctcaagt gattcatccg 15660 ccttggcatc ccatagtgct aggattacag gcgtgagcca ctgcactcag ccaaaaataa 15720 taagtggtta gtgaatctgt tgttggttca tgtactttta aaaattgtct tccactgggc 15780 gcagtggctt atgcctgtaa tcccagcctt tgggaggcca acgcgggtgg atcacctgag 15840 gtcaggagtt tgagaccagc ctgggcaaga tggtgaaatc ctgtctgtac taaaaaaaaa 15900 aaaaaattag ctgggagtgg tttgcatctg taatcccagc tactcaagag gctgaggcag 15960 gagaatcact tgaacctagg aggcggaggt tgcagcgaac cgagatcgcg ccattgcact 16020 ccagcccgcg caacaagagc gaaactctgt ctcaaaaaaa aaaaaaaaat tgtcttccat 16080 gaaaaacagc atggatgatt tatgagcttt agtttaagcc tgctttgttt gtaatttcat 16140 gttactacag tggttatgat ggcctgtatt caagaatgtt gagtctgtgg gtcatttatc 16200 ttatagctga ttattttata gtgggttgga aataaggttg tgggatttct gaatccaaac 16260 cagaatgctg agaggacatt ggtaataaga tagtgtcctc agtggtgcat gcctgcagtc 16320 ccagctactc gggagactaa ggtgggagga ttacttgagc ttgggagatc aaggctgcag 16380 tgagccgtga tggtgccgca ctgcagcctg gacaacagag caagaccttg tctcaaaaaa 16440 aaaaaagatt ttaaaaagta tcctcaaaga ttgcttttct tcaagttaat ctgcaaattt 16500 ttggattcta ggacagtatg agatgttaac ttcccacagt tacttgtgat aaggtcttac 16560 taagagaatc gcctcattct agttttaacc ttgttcttgg aagtttatac caaatttttg 16620 tttgcttgaa attcattcat ttcagtctat accatttgac tgaatttcag aagggttctg 16680 ataaatcaaa accagtgtgg tactaattac attctttttt ttcttttttg ggatggagtc 16740 tcgctttgtc acctaggctg tagtgcagtg acatgatctt ggcccattgg gttcaagtgg 16800 tgcccctgcc tcagcctcct gagtagctag gattacaagt gtgtgccacc attcctggct 16860 ttttagtaga gctggggttt cactatgttg gtcaggctgg tctcaagctc ttgacctcaa 16920 atgatccgcc tgccttggcc tcccaaagtg ctgggattac aggcatgagc cactgtgctc 16980 ggccctaatt acattctttt aaaaacttta tcttaatttt tttcccagat gttgaccata 17040 ttagctaaga aatttgaggt cctggaaaaa tgttttaggg gaagcaaaag gtttggtatg 17100 catgttttgc ttttgcttgt ctttcagttc ttaattcagg tgatatgaga ctttacagaa 17160 gtcaaaatgc cagagtaaaa aggtggttat taaaaataaa aaacctcaaa tgtcaaaatt 17220 aagttactta aaagtctggt tctaccattt actttgagta ttaaactaga cagttgagat 17280 tgtataacca tctttgaaaa tgatggttgc taaaggtctg gaggctctgg aggtgatcca 17340 taaagccgct cttattaggt gctttcagga tttataaggc ctaagtcctt gtgatagcac 17400 agcttatctt tattttgttt ttagtttttt tttttgagat ggctgtctac caggctggag 17460 tgcagtggta tgatcacggc tcactgcatc cttgaccgtc ctggggtcaa gtgatcatgg 17520 ctgactgcat ccttgaactt ctgggctcaa gcgattctcc tagctcagcc ttttgggtac 17580 ctaaaactgc aggtgtgtgc caccacagat ggctaatttt ttattttatt ttctatagag 17640 aggggtctca atatgttgtt gcccaggctg gtctctgaac tcctgagcat cctcccgctt 17700 ttaccttcca aagtgttggg attacaggca tgagccgctg cacttgccaa aatgatcttt 17760 tttttttgga cagagtcttg ctctgtcact ctggctgtag tgcagtggag cgatcttggc 17820 tcactgcaac ctccgcctcc caggttcaag ctattctcat gcctcagctt cccaagtagc 17880 tgagattacg ggcctacacc acaacaccca gctgttttgt gtttttagta gagacggggt 17940 tttaccatgt tggccaggct ggtctggagc tcctgacctc aagtgatcca ctcgcctcag 18000 cctcgcaaag tattgggatt tgggattaca ggcgtgagcc actgcaccca gcccaaaatg 18060 atcttttata tgaacatctg tagcttttat tgttctactg ttctggtgta gtctgtttta 18120 ttgttgtaac tatctgagag ctggtaagtt gagaccaaaa caacaacaac aaaacaacca 18180 aaatcatgct taaaactggt tctttccaag gttgcatttt tgttaagatc tacttaatag 18240 ttgattggat gatttttttt tcccccctcc ggtctgaggg atggttgctt ctactgtata 18300 ccactgttat cgccagtatt taattatatt tgttctggtc tgtgaagttt ggtaggaagg 18360 gaagaaagag ctctggttgg cttttctaga ttgacaagtt agaaatttca cttaagagtt 18420 aactgttctt tttattttaa aggacaattt gataatgtca acatttaaaa accaaactac 18480 tagatacact agttaagttc acttcatagt atcttctctc aggaaatgtg cctgcatatg 18540 tatacagagg agaaaatacg aggatatata ttgctgtagt ttcacacccc tctgcccttg 18600 taattggtca ggggaatact ctgcctccac tatgagaaaa taaccagctg tctcaataaa 18660 tgttatgtat tattatatat tgttagaaaa gcaggctgct gaacaatatt gtttctagtt 18720 taaaaatgca aaggaaaact attgtttctg tagacataca gtatgtgtgt aaactttgta 18780 aacaaagttt taaaggatat acagacactc cccaattttt cgacttaatg atggtgtgaa 18840 aatgatagct attcaatata cccctcagtt tatgatggga ctacatccag ataaatatat 18900 cataacttga aaacattgta agctgaaaac acactgttga ctcgattata tttacgatag 18960 gattatctgg atgcagctgt gttacaaatt gagcatctgt acatcaaaat tttattggta 19020 actttgccca aagggggtgg gattgtgtag ggtgctcaaa gggaacattt cattttatct 19080 gtgccgaaat tttttacaag aatgtattac taatttgtat agtttaaaaa atagataaca 19140 ctgtttatcg gtgtgatttg cctaaattga aataaaagca acaaatagca tttagactta 19200 attttccttc atgatttgaa tagaaaattt gagtcagttg tagtaaggaa cttcaggcag 19260 tcctgaaagt caacttttta ttattaatac agagaatagc tttgcagata aacagatatt 19320 cacagaagta tattaacatt taacttctga tagctttaaa tgtgaaaatt acatcatggc 19380 aagatttaca tggtatataa ccatctgttt gcttatgtca tgccctttta aaacttctga 19440 ataacttttg caagtttctt ctcagtgtag gacttatttc atttagttat tttcattaat 19500 atatgatagg tgttaaaaaa acactagatg tggtattaat gccagacaga cttggtaatg 19560 gaataagcct agtgaatttg tgttttttag tgatagtaaa tgggaagttg gaattaccct 19620 gcagagaaaa atttcaaaat gaagtaggat ttgataataa tgggttgtac atatttctta 19680 ataggtatct caggccagta tagtggctaa cgcctgtaat tcagcacttt gggaggcaga 19740 ggtgggcaaa tcatttaagc tcaggagttt gaggccagcc tggacaacat ggtgaaacct 19800 cacctctaca aaaagtgcaa aataggcata gtggtgcaca cctgtggtcg caattgttca 19860 tggagactga ggcaggagga tcgtttgaac ctgggaggtg gaggttatag tgagccgaga 19920 tcatgccact gtactccagg ctggtctgga actcctgggg tcaagccatc cacccgcctt 19980 agcgtcccaa agtgcagtgg ttcttaaact tggttgcaca ctgggaaaag gaggggtggg 20040 ggtgcttaaa aattattgat ggcttgattc cagtctccaa aattctgact ttattagggg 20100 aaggaatggg cattggtgtt tttgtttttg ttttgagaca gagtcttggt ctgtcaccca 20160 ggctggagtg cagtgatgcg atcttggctc acggcaacct ccacctccca agtttaagca 20220 attctcctgc ctcagcctcc caagtagctg gtactacagt catgcaccac catgcctagc 20280 tagttttttt tatttttagt ggagatgggg tttcaccatg atggccaggc tggcctcaaa 20340 ctcctgacct caagtgatcg gcccaccttg gcctcccaaa gtgctgggat tacaggtgtg 20400 agccaccgcg cctggctggg cattgggatt tttaatgtgc agctaagatt gcataccaat 20460 gctttgaacc cagtccttgt catctgtggt tgtcatggct cattagcaac aattctctgg 20520 taatttgttt tggtatctta tgtaaagtgg tctctcccag ttttcctctc tctttccttt 20580 gtgacactta acacagtgta cttttggctt ggttacttgt ttgtggtcct tctcccctct 20640 acggtagtgg gaatgagcca tcttgttcac cactatatgt ccagttgcta gcatggggct 20700 ggtacagatg gttgagcaaa tgattttctc acaggtatga gccacagtgc ttggccaaaa 20760 acatgttttt aaaaagtcaa acagggctga ggtgaggtgg cttatgcatg taatcccagc 20820 ccttgggagg atgagggtgg aggatcacgt gagaccccgt ctctacaaaa aagtttgtga 20880 aaattagcca gccatggtgg cgtgtgcctg tggtccccac tactgaggtg ggaagattcc 20940 tgagccaaga agatccaggc tacagtgagc tatgatcata ctactgcact ccagcctggg 21000 tgacaaaatg agaccctgtc tcaaaaaaag aaaaaaaaag aatctttgag tgctgcattg 21060 tagttagaac tctgaaggta aggaagtaga tacctgaatg tcctgttcca ttttcattgg 21120 ttttatgtga cagctaacaa ttagtattta attagatggc tatttgatag gtttttaaaa 21180 aactcctggt attgatgata ggaatgcata ttttttccct aggtaacagg gtgagatagc 21240 taatggatta taatcatatc tcacttattt taatgggttc ataaatgcct gcaattttat 21300 cttagactga gtctgcccct taagctacat taagtgtaag taaacaaatc tttgttaata 21360 ttgtttaatg ccaaatgtat tgccttattt ttgtctccca tctgtaggct aaagaaatcc 21420 tgacaaaaga atccaacgtg caagaggttc gatgtccagt tactgtctgt ggagatgtgc 21480 atgggcaatt tcatgatctc atggaactgt ttagaattgg tggcaaatca ccagatacaa 21540 attacttgtt tatgggagat tatgttgaca gaggatatta ttcagttgaa acagttacac 21600 tgcttgtagc tcttaaggta atttcaattt tatgttgggg catgttgaaa tgggtaagac 21660 agtcctcttg aaagtttttt tccccccagt tattttctct atctgaatgt taaaacaaaa 21720 ttccacattt aggaatgcat atgttcaggt tttggactta aaaatcatag gcgtctgcgt 21780 tctgagtaag gggatggtac agaatcaaaa caaaaggagg agaatgaatg cctcagtcag 21840 attgtttgaa aaaataggct gggcgtgatg gcttatgcct gtaatcccag cactttggga 21900 cgctgaggta ggcagatcac ctgaggtcag gagttcgaga ccagcctgga gaacatggtg 21960 aagccccatc tctactaaaa gtacaaaaaa actagccggg ggtggtggcg ggtgcctgta 22020 atcccagcta ctctggagcc tgaggcagga gaattgcttg aacctgggag gtggaggttg 22080 cagtgagctg agattgcgcc actacacgcc agcctggaca acagagcgag actgtctcaa 22140 aaaaaaaaaa ggaaaagaaa aatgttagag ggctggtcaa atagatattt tagttcagta 22200 aattggggta tggagaggta atataattag atttgtgttt ttagatactt ttgaagtatt 22260 ataaatatat aaacatacca aatgtcaaaa tgttttaaat cagctggtca cggtggctcg 22320 tggctataat cccagcactt tgggaggaca ggacaaagtg agcatattgc ttgagtccaa 22380 gagtttgtga ccagcctggg caacatagtg agaccttgtc tacaaaaaaa taataataat 22440 taaccgagtg tggtggcaca ggcctgtagt cccagctact caggctgagg tgggcagagt 22500 tgcttgagcc caggaggtcg tggctgcaat gagacttgat cttgccaccc tacactctaa 22560 cttggggaac agtgagaccc tgtctcaaaa aaaaaaaaaa aatatcgagg aagaagttca 22620 agaaaaaaaa gaatttttct gagaaattca agaaaacatt tgctgtaaat atttaacaga 22680 gaatattatg tacattacat acattatgta tgtacatatg gtttagctca gttgaatagt 22740 ttccagccat taaccatgac agtaataatc tagaataggc tgggtgcagt ggttcacgcc 22800 tgttatctca acactttggg aggctgggga aggcgggaag gatcacttga gtccaggagt 22860 ttaagaacag cgtagtcaac ataaacccca tctctacaaa aagtttaaaa aaatagccag 22920 gtgcagtggt gcatgcttgt agtctcagct actcaggaag ctgaggtggg aagatcactt 22980 gaacctggga ggtcaaggct acagtgagcc atgatgcacc actgcactcc agccgtgtga 23040 cagagcaaga cccctgtgtc tttaaaaaac aaaaatctag aatggtagaa atgataaccg 23100 aaaaaagcaa gttccaaaat tacatgcatt acatgccaat tgtagctttg caaagtgtac 23160 cgatgatgat ctgaacatga agcaaaagat ggtaaagagc tgttagggtg gcaggcttcc 23220 gcctttccct tgaaataaat tgtttgactt tacctttacc caaaaatctt ggggagattt 23280 ttttccccct aggcaacaga ctgattttta ttattaaaga atattcaaaa tgataaatgg 23340 ctgtcacaga ctttcctaat attagagaag cagtgagatg tggttttagc gtgagctgaa 23400 ttttcttgat taatatctga attgtgtaaa gtcctacgaa atatttttgg agatcgttat 23460 tttctgcatc tgtaaaatga aggagttgga ctagatggtg gaaggtttct gcggtgactc 23520 caaatactat gaaaagagct aacctgtgct aaaaacccag gatgaatccc agtgctcaga 23580 ggaatgaatg tgctaccttt aagagtatgc aagcagcctg ggcaacctag tgagacccca 23640 tctcttcaga aaacaatttt agccagacat ggtgacatgt gctgtagtcc tagccactct 23700 ggaggctgag gtgggaggat tgcttgagcc taggaatttg aggctgcggt gagctactcc 23760 actgcatttg agcctgggcg acagagtgag atcctatctg gggggaaaaa aacttactca 23820 tggaggttaa tgggctgaat tgtagccccc aaaattcatt tgtctaactc ctaacccaca 23880 gcacctcaga atgtgactgt atttggacag gtaattaagg taaagtgggg tcacatggtt 23940 ggaccctaat ccagtgtaag tggtatcctt attagagatt aggacacaga tggggagaag 24000 ataagacaga tggagaagat gagcatccat aaatcaagga gagaggccat cagaagaaac 24060 caactgtgct gacaccttga tcttggactt ctgcctctgg aacagtgagg aagtaaattt 24120 ctgttgttta agccacctat tctctgatgc tttgttgtgg cagccttagc aaaagaatac 24180 cgggggaaag gatgtttctt tcagtaaatg gtgctgaagc aaatggatca gaatgtatca 24240 tagaactaaa tgtaagagct aaagctacaa aacttctaga ataaatcaga aaatctttgt 24300 caggttagtt aaagattctt agatataaca aaagcacagt ccataaaaaa ttgaaaaatc 24360 ggactttatc aaaattaaga ttatgttttt tcaaaagatg acaccagaaa aaaagtactt 24420 gtattcagat atttaaaaaa aaaaaacttt tacagctcaa taacaagaca gataattgta 24480 aaatgggcaa aatatttgaa tagatatttt acccaagaag atgatacatg agtggtcagt 24540 gagcacatga aaagatgctt gatatcaatt gtaatcaggg aaatgcaaat taaagtaact 24600 gagttactac ttcacatcta ttagaatggc agtaatcagt aaggttgata ataccaagtg 24660 ttggttaata ccggagatta aactcttcat gcattgctgg tgggaatata acatggtcta 24720 gctactttga aaaacagttt gactgtttct caaaaagtta aacgttacca ggtgatccaa 24780 caattatatt cttaggaatc ttcctaaaag aaatgaaagc atatgtccac agaaaaactt 24840 ccatgtgtat gattatagca acattattca tagtagccaa aaacctcagt aacataattg 24900 ccagttcatc ttaactgtga atagagaaac aaaaaatggt atagccatac agtgaaggac 24960 tgtgcaacta tgaaaaggaa caaaccactg atctatgcca gaacatgggt gagcctcaaa 25020 aatattctaa gtgatattta gccaaaagac tacattttgt attccattta tatgacattt 25080 ctagaaaaga caaactcaag acagaaagta aatcagtggt tgccaggagc tggagtggga 25140 gtggagagtg actgcaggag ggatcttttt gggataatgg aaaggttcta aaactggatt 25200 gtggtgattg ttgcataact ataaatttgt gaaaaattac tgaaccgtaa agtaatttat 25260 ggggttttaa tagcccattg atttatgttt ggtataaagt tggtggtaga gggagtttga 25320 ttttagttga gatgaggtgg gcaaacaccc catgggcatt taaaaattct gtagagctgt 25380 ggttttggtt cactatgata tgaggatgtg tgctgttttt tcaacaaaat tgtaactgca 25440 tttttttcta tagaaagttt cttgaatttt cattggtttt tgaaagagct agttgtttat 25500 gttttgcatt ttattgtttt aggttcgtta ccgtgaacgc atcaccattc ttcgagggaa 25560 tcatgagagc agacagatca cacaagttta tggtttctat gatgaatgtt taagaaaata 25620 tggaaatgca aatgtttgga aatattttac agatcttttt gactatcttc ctctcactgc 25680 cttggtggat gggcaggtat gtggatctaa aactcattgc tgattatttc agagaatctg 25740 ctttctttag tgttccggtg cttatgactc tttccactct taacacccct ttggggttat 25800 tttttaatag atattgatag cattcattca aacttaaaaa aaatttaatt tgacaaatgt 25860 gtccaacttt tttaagtgca ttattcttta tatcaggcac tgaactgtat ggtgggatca 25920 caggggtggg tgatgtagcc catttaccct ctcagactgt ttttggacaa gtctgggaga 25980 tgtaaggagc taattattgg cctaggtaga tgtgcacaca aaagaactta caataaaatt 26040 ttatgagtat gaagatacta gtctctggca aagggcagga gtgatatcca agaagttttc 26100 tcaaaggagc tgtttgaagt ggagtctggt tgtgacagcg atggtctatt aggaagaaca 26160 ggagtgtact caatagttca gagtgggctg gagcaaagat gaaccctgca aagtcaagtc 26220 tcattaaggc agagcacaga aacaccagag gggccaagtg atgggggtgc ccagctcatt 26280 ttttaaaatt tttgtagaga cacagggtct tgctgtgttg ctcaggttgg tctcaaactc 26340 ctggcctcaa gcagccctcc tgcctcagct tcccaaagtg ctggggttac aggcatgagc 26400 cactgtgccc agccaagaat gtttacaaat tttttaggtg aggttaacct tttaaaccac 26460 tcttttcaga tcttctgtct acatggtggt ctctcgccat ctatagatac actggatcat 26520 atcagagcac ttgatcgcct acaagaagtt ccccatgagg tatgactttt atttgataaa 26580 attctttcag aaaacttatt agggagtgga gaagtttaat tgtaagttca ttagatgtat 26640 tctctcatat aattctcaaa aacaacttga ggtgtgagca ctcttgtctg ccctgttgtt 26700 caaaagaagc tgacattaag aaacctgcca agatcacaac agctgtgaag ctgggcttat 26760 taggacctgg gttgtctgtc ttagactctg aaatttgcat aatataggca gattaattag 26820 ggcatgttgc tgctgggtcc catggatgtc agctgtgtct cataaagtct gctggaaaat 26880 acatcttatt tgatgcagat gtttgacttc aactgcacag tctattagca ttgtccacaa 26940 acatcactca aaaagggtgg cttagggtgc tgcttctgat gaattactca aataatgtgt 27000 taaccatgtt tgtaaaaggc attttataac ttgaagttat tttttagggt ccaatgtgtg 27060 acttgctgtg gtcagatcca gatgaccgtg gtggttgggg tatatctcct cgaggagctg 27120 gttacacctt tgggcaagat atttctgaga catttaatca tgccaatggc ctcacgttgg 27180 tgtctagagc tcaccagcta gtgatggagg tatgctgtgt tctctgaaat atgacttgtt 27240 tttttagtaa acttaaggaa agaagatatt agggagtttt aaattaatga catttgtgag 27300 aaattccatc tattccttct gatgagttta tcattatagc ctgagccttt tcagtagatg 27360 cacactacta gaattaaaag ctcaagcttt tgattacaat gaatccatta acatgttttc 27420 agagaaatct gaatgactag gaagtacttg agatttaaga gtgggaaagt aagatatagg 27480 gagttggtgt gcatgtatga tattttatga tcccaatgtt atttcgtata tttaatgtat 27540 gtatgtagga gagactggaa agaaatacac gaaaatgttt taagtggtcc tttggatgct 27600 aggattacag gtgatttgaa agaagggaaa gaggcaaagg ttcttgacat ggcacactga 27660 ttagaatatt tttaaaataa ggtcctaatt agattaatac tagtacaact ctgtggttat 27720 caataaaatc tgactttgct aaaggtaaag aaatttgtga acaatactaa acaaaaagta 27780 acaaaagtag ctcatgcatt caggtggatg gactttgagg aaatgaggct ggagatccta 27840 gaggcatatt gcatttgttc ctgggcagtg accaacttaa caggccttta cctgtgactg 27900 acaaaattgg taaggttctg gtctccagga ttaagtatct acttctctag ggaatggcca 27960 cttaaatact tattcttaaa tcatgtttta aggccactgc aagagaagag gctaatgaga 28020 cattgtcatt tacagcaagg cagatacatg agaagtattt cattctttgg tggttgctgc 28080 tgtttgttaa agcctttttt ttttttaaag tactaaaaaa aaaacaagtt ttgtaattag 28140 ggggaaaata aatacatgtg taccttacag gttgtataaa gtacaggaga ttattaagtc 28200 atccagaaaa aaagcattaa catttcttaa gcccctttgg caggggtggg aatgagtaga 28260 gatttttttc tctcttgagt gaagtaacat ttagttgaat tgagggagta aagtaagatt 28320 tttgtctcct tttccttgca gggatataac tggtgccatg accggaatgt agtaacgatt 28380 ttcagtgctc caaactattg ttatcgttgt ggtaaccaag ctgcaatcat ggaacttgac 28440 gatactctaa aatactcttt gtaagtaatt ctacctgaac attttcttgc attactgaat 28500 atgtagggtt ttggtttatt tattttgaga gagagggtgt cactatattg cccagtcttg 28560 tctcgaactc ctaggctgaa gtgatccttt caccttggcc tcctagagtg ttggggttac 28620 aagtataaga tacagtgccc agcctgaata catagtttaa atgtgaggtt taagagttta 28680 agggggccag gcgcggtggc tcacgcctgt aatcccagta ctttggaagg ctcgggtgga 28740 tcacctgaga tcaggagttc gagacaagcc tgaccaacat ggtgaaaccc cttctctact 28800 aaaaatacaa aaattagccc agcgtggtgg cgcctgcctg taatcccagc tactcaggag 28860 gctgaggcag gagaatggca tgaatccggg aggcagaggt tgcagtgagc caagatcgcg 28920 ccactgcgtt ccagcctggg caacagagcg agacttcgtc tcaaaaaagt gtttattaag 28980 ggattatgct atgtcagatt ccaactttta tttttaagtt ttgttttttt tttaatggat 29040 ctacattgaa acacaccaac aggaacttat tatatataga tcttcgtatg aattactagg 29100 tttttcactt ttaaaaccat gttttgtgtt tttttgttgt ttgtttgttt gttttttgag 29160 acggagtctc actctgtcac ccaggctgga gtgcagtggt gcgatcttgg ctgactgcaa 29220 gctctgcctc ccgggttcac accattctcc tgcctcagcc tcccgagtag ctgggattac 29280 aggcgtctgc caccacgccc ggctaatttt tttgtatttt tttagtagat atggggtttc 29340 accgtggtag ccaggatggt cttgatctcc tgacctcgtg atccgcccgc ctcagcctcc 29400 caaagtgctg ggattacagg cgtgagccac tgcgcccggc caaccatgtt ttgttttgtt 29460 ttgtttgaaa tttcagtcaa ggtaaaaacc atgtttttat aaaagcatag tctctgtggc 29520 tgaagttgcc ttgactcttt caaggggagt acagttaaca gaccattatc attaaggttc 29580 tgttacatta agacaagcca aaattgaata ggaagtgtaa agtttggaag actcatgttc 29640 ttgatcttga ttgtcactac atggaggtcg tggaacataa accatgaatc ggtcttgttt 29700 ttcagcttgc agtttgaccc agcacctcgt agaggcgagc cacatgttac tcgtcgtacc 29760 ccagactact tcctgtaatg aaattttaaa cttgtacagt attgccatga accatatatc 29820 gacctaatgg aaatgggaag agcaacagta actccaaagt gtcagaaaat agttaacatt 29880 caaaaaactt gttttcacat ggaccaaaag atgtgccata taaaaataca aagcctcttg 29940 tcatcaacag ccgtgaccac tttagaatga accagttcat tgcatgctga agcgacattg 30000 ttggtcaaga aaccagtttc tggcatagcg ctatttgtag ttacttttgc tttctctgag 30060 agactgcaga taataagatg taaacattaa cacctcgtga atacaattta acttccattt 30120 agctatagct ttactcagca tgactgtaga taaggatagc agcaaacaat cattggagct 30180 taatgaacat ttttaaaaat aattaccaag gcctcccttc tacttgtgag ttttgaaatt 30240 gttcttttta ttttcaggga taccgtttaa tttaattata tgatttgtct gcactcagtt 30300 tattccctac tcaaatctca gccccatgtt gttctttgtt attgtcagaa cctggtgagt 30360 tgttttgaac agaactgttt tttccccttc ctgtaagacg atgtgactgc acaagagcac 30420 tgcagtgttt ttcataataa acttgtgaac taagaactga gaaggtcaaa ttttaattgt 30480 atcaatgggc aagactggtg ctgtttatta aaaaagttaa atcaattgag taaattttag 30540 aatttgtaga cttgtaggta aaataaaaat caagggcact acataacctc tctggtaact 30600 ccttgacatt cttcagatta acttcaggat ttatttgtat ttcacatatt acaatttgtc 30660 acattgttgg tgtgcacttt gtgggttctt cctgcatatt aacttgtttg taagaaagga 30720 aatctgtgct gcttcagtaa gacttaattg taaaaccata taacttgaga tttaagtctt 30780 tgggttttgt tttaataaaa cagcatgttt tcaggtagag cttaaactaa atgatgtgtt 30840 tacttagtgc agtttctggt tatgaatatt atattgctat gtgtatatta tatggactct 30900 ttaaaatgat tgacagattg gcaaattctt aaatctttgt acattgttga gtcatatgtt 30960 cttagagtta aatttgtctc agataagaaa gtgttaaagc attagcctgt gtcaagttct 31020 ttgagtgata ctagtgaaac caaatagaaa actattgttg gatcatgatt tagtcttatg 31080 tacattcacc cgaagacaaa aatggtactt aaagtggcag tgttcaacat ttaatgagtt 31140 tttccccttt tatccttcga ataggattag atgtttaaaa aaaagttctt ctgtgggaac 31200 taatatttga tattttaacc taccagagta aacaggaaca cttaatcata cttgtgagtg 31260 tagtaaataa aagttttctt gctttgtgct gtgttgaatc tggaaccaac agggaagtta 31320 tagcatatcc cctttctaaa atgcttgagg aacacataca taccgaatgt cttttctgat 31380 ctaattgata gtatttttag tggcttgtgg agttaatttt ccaaagcaaa aggccattag 31440 ggtttctaca tttcatttca tttcattctt ttctttcaca agaaatacat tctctgtgtg 31500 tctttttgtt gctctgtcac tctatgccct ttctctccga ctgaacaaat agcttatcca 31560 tgtgcagtgg ttttaatacc caaacaatct agacaccaag cagctatttt ttccggtcct 31620 gtgatatcag aattgaccaa ggaatacgta tattgtaatt gacacgtggt ggtatcttcc 31680 aggtacaaat tctctaaatt ttgtggttag cagaatggga cttgtgataa gaatagcttg 31740 gttttagcat aactagggtt taaaataatt gtttaattat agagactgac caggctttgg 31800 ccatctaaac tggaaagtgt tagtacccta ccttcttttg aaaatggcta tggtaaggaa 31860 aatgtgttag taaattatgt attttcttga aaaatacata attatggttg gatgggaatc 31920 actaagttgg gtgttaactg atgtctcaat tagtaacatt aggattttca ttaataaacc 31980 taaaaagctt tccctaagaa caggcctggc acagtggctc atgcctgtaa tcccaacact 32040 ttgggagacc agggtgggtg gatcccttga ggtcaggagt tgaagagcag cctggccaac 32100 atggcaaaac cccatctcta caaaaaatat gaaaatcagc cgggcttggt ggcataccat 32160 agtcccagct acttgggagg ctgaggtgag aggatcgcct gagcccagga gacggaggtt 32220 gcagtgggct gagattgtgc cactgtactc cagtctgggt gacagagcca gaccctgtct 32280 caaaaataaa gaggattctg agtttgtata gtgagggctt gcagaaattt tgaaacttat 32340 tttgtaagtt tacaatgaat ttgtacatga tgtgctcatg tcttgggttg agtatcctag 32400 acatgatttt ttcatttgct gcatattaaa catttgttgg ttgtagtcgg tatttcttaa 32460 atagaagttt gtcaatatta gattagtttc aagaaggact tagctcagga aaaggatagt 32520 tatttctgtg gttctcagct ttgatgccta cagagattct gatttaaatt gctggaggag 32580 ggcccaggca tacatttttt aagtttccca agtgattcta atttacatct agggttaaaa 32640 acagccaggc gcggtggctc atgcctgtaa tcccagcact tggggaggcc ggggtgggtg 32700 gatcacctga ggtcaggaga tcgagaccag cctggccagc gtggtgaaac cccatctcta 32760 ttaaaaatac aaaactgacc ggtgtggtgg caggagcctg taatcccagc tacttgggag 32820 gctgaggcag gagaatcact tgaactcagg aggaggaggt tacagtgagc cgagatcgtg 32880 ccattgcact ccagcctggg caaaaagagc aaaactccat ctcaaaaaac aaaaacacct 32940 cactgctgtt tcctaagtac atacttaaga aaattgggat acatggtggt ggttcatgga 33000 tgttgataag gaattaaaat gtaccgtgcg actctctgtt tcagtggtga cttttacctg 33060 tttagtataa atattccttt gcttccaacc ataaatgtgt tcttagaaat gggcctatag 33120 tttagtaacc tatagtttgg taataggctt gtttgttttc agatggattt tggttctgtg 33180 agctaaagct attttgcatt aaagccttcg tcctcacaca ttgttttgac atatttctag 33240 tcttcataaa cttttttaat ttagattttt ttcccttcac aagtatacat ctgttttagc 33300 aaatagcctt atgaaggttg tagatgtatt attttgggca tgcctggtga tttctatatt 33360 ttttccaatt acatttaaag ctttatgttt taggaatata agtacatttt atttctactt 33420 tttattatat atatttaatt gcacaagtac tactgtctag aaaaaaatgg gatgttgcta 33480 acacagcatt gttggcttgt aggcagtgct gtcctgtaaa tagattgaaa tgtattttta 33540 tcagctggta tataaatttg aggaaagaaa aatgtggact gtgtttgaat tgtttaaaag 33600 ttgaacatac aaagatcagt ggtaaccaag taagtgatat aggcaacaga ccccagtttg 33660 ttttgtattt gctgtcatgc ctggaccaga attcctcatt cccaagacgt gaaagaggaa 33720 ataattatag ctaaatgagt gaatgctgga aaaagtcaca cttttttgtt ttttaaggaa 33780 aagcacaaac cctcatgtct gaggttgaat ttaattaagt gcagtcggcc ctccatagtc 33840 aagggttcca catctgagaa ttcagccaac catggactga aaatattttt gggaaaagga 33900 accaataaaa accaatacag tggccaggag cagtggctca tgcccataat cccggcactt 33960 taggaggcgg aggcaggggt ggggggatca ccggaggtca ggagttcaag accagcctgg 34020 ccaacatggt gaaaccccgt ctctactaaa aatataagaa ttagctgggc atggtggcgt 34080 gtgcctgtaa tcccagctac tcaggaggct gaggcaggag aatcacttga atctgggagg 34140 tggaggttgc agtgagccaa gatagagcca ctgcacttca gcctgggcaa cagagcaaga 34200 ctccgtctaa aaaaaaaaaa aacacagtat aactatttac atagcaattt cattgtttag 34260 gtattatgag taatccatat tttaaagtat agccagccat caagtatcca cagagtttgc 34320 atcaaatgag tcaatggagt caaccaacct gggattgaaa atgcagtgtt cttttttttt 34380 tccccctgag acggagtctc tctctgtcac ccgggttggg gtgcagtggc acgatcttgg 34440 ctcactgcaa cctgtgcctc ccgagttcaa gcaagtctcc tgcctcagct tcccaagtag 34500 ctgggattac aggcgcggct cactacgccc agttaatttt gtatttttag tagagacagt 34560 ttcaccatgt tggccagggt ggtatgaaac tcccaacctc aggcgatcca cctgcctcag 34620 cctcccaaag tgctgggatt acaggcatga gccaccgcgc ctggccaaaa aatgcagtat 34680 tcttgaatgc agaacccact ggtaaagggg actgacttcc agacttgact atccaaggga 34740 ggtgtcctgg atccagtctc ccaagattac tgagggatga ctagtttgta tcctcaacag 34800 gcttgtgaga agtagccagg attttgtcta ctctgcgtac gatgctgcta atgctgcaaa 34860 ccactgctct tattttacta cttgggtccc ttcggaatct attttgagat agcctcagcc 34920 atgatgtaca atcttctcaa ccattcagtg ttgttagtgg gaaggacttt aatgtgttct 34980 tcctgctggg cacagtggct cccgcctgta atcctatcac tttgggagtc cgaggtgggt 35040 ggatcacttg agcctggagt tgaagatgag cctggccaac atggtgaaat cctgtctcca 35100 ctaaaaaaac aactagctgg gtgtgatggc acttacctct agtaccagct agttgggagg 35160 gtgagactaa atcgcttgaa accgggaagc agaggttgca gtgagctgag atcgcgccat 35220 tgcactccag actgcgagac tcttgtctcg aaaaaatata tttttaaaag ctttcttcca 35280 agaaataatt tagctgttct tccttagtgc agtggtaggc tgtagccacc agcaagcaag 35340 attactgatg gtggaagtca aaggccagaa tcgtgatagg aagttaagag tgcttgctcc 35400 atagaacact atggaagacc acaatggaat gtctaattaa agctattaga aatgcccaaa 35460 gctgttgagt gcctctacct caaggcttag cataccttct gatctgcaag gagactaatc 35520 attaggcttt ttgcagttct agaactgctc tgagtatggc attggaacct ggggatagga 35580 gcaagagcta gtaattgggt ttggcaggcc tggtcaaagg ggagctttaa ggtcaggtat 35640 atccaaacat atcaggaagt aggttacatg gtgtggtgaa atataccaga tgtaaaatct 35700 gcagaatgta ctcctgctgt tgagtccaac ttgcaatgta agtccctttg cagtgaaggt 35760 cttgagtatg tattgcatgc agcaggcaga ggagcgggag gagaaactgt ataatgtatt 35820 tggtcttgaa catcctttgt tttccaaaac cacattcatc tctaccttgg ttgaaatgta 35880 taaaactacc attttaagtt tcatcttttc attgatctat gactaaaatt aggatgcaaa 35940 gacaaaccca aggcttagcc cactgctcct cctcagagct gcactggaac tggttgtagg 36000 gtaaagggat gtaggcaatt taaatatgag tcaagatttt tgggatggca gtatggctct 36060 catggcctct ggaaggatcc aggtgatgtc cgacctggta tggcctcttg gcctgttgcc 36120 accgcaccag gttttgccct taattttagt cttggctctg gatttcttta gggtattgtg 36180 atagctttgt tggtctttgg gtttttctag tttcaccaca gatcctagga aaaataacca 36240 tactctagaa atgagaagcc aggattttta aagtagggct gggaatatga gtagacatct 36300 tttggatttt caaaatgtat ttgcttaaac caccttttgg cagcagttgg acatttctgc 36360 tatctacctg agtggcagat ggcctacagc atttggtatg ttaaaagagt atagagtaag 36420 tgattcatgg cttctgaaat acatcaattt gacgttgcag tcttgaaaag gcaatagctg 36480 acatatcctg aaccagcttg ggtttccagt gcactcagtg ctgaaataat tgctgtgaag 36540 ggttgagttg aattcagaat gacttaggac agtagtagct actgaatgtt caatctttga 36600 aatgacagaa ttagcttata gtgaatattg tctagcactg agcatttcca tgtgccaagc 36660 atgttacatg aattattgag ttttctcaac tctgttgaga agagcagaga aggttctgct 36720 attcaggcat tctgatcgca gagttcctga ttttcaggaa gcattaggtt tctcttgcct 36780 ttgctgaaat tcttattagt ggtgtttgaa gggtgggcaa tagcctacac agctttaggg 36840 tttagatgtg tcgcttagcc ccattcagga agctaggtgt gtgatgggcc ctttggggag 36900 tgtgtggtga ggaaggcttg catgctgggc cttttgagtg ttggcccaag tcaggtagcc 36960 tggttacagc attctcaaga gttcatggac tgccgggcac agtggctcac gcctgtaatc 37020 ccagcacttt gggaggctga ggcgggtgga tcacaaggtc aggagttcaa gaccagcctg 37080 gccaagatgg tgaaacccca catctactaa aaattaaaaa aaaaaaaaaa ttagccaggt 37140 gtggtggcgg gcacctgtaa tcccagccac tcgggaggct gaggcagaga attgcttgaa 37200 cccgggaggt ggaggttgca gtgagctgag atcgcaccac tgcactccag cctgggtgac 37260 acagtgagac tcgtctcaaa aaaaaaaaaa aaaaaaaaaa gagttcatgg actaaccttt 37320 gtgtctcaag ggcatttatt tgagccagag tttgaagggg agttaggtga ggttcagctg 37380 aaccaccaca gcctaacaat tgctcttgaa gataaaagaa ttttcacagt ggacccgcat 37440 taacaaatac taaaggaaaa ccagaaagta tatatggagt ggagcacccc atccctaact 37500 tttaaaaatt catctaagtg gcataattcc tccatttttt atctgaattc ccaactgaat 37560 tgtgggtttt cttgaatgac ctgagttttt tagactcttt tataaacttc tgggattctg 37620 tacaagacta ctggaaacta gtcagctctt taggctgaca gttgtagtaa tgaatgaaat 37680 ctcatgctag cctgtcatca cctgaccatc ttcccctcct tctgaagacc cttaccttct 37740 aatctgagag gagccagaag ccaagaccaa cttactttct tgtctggtgt cctgatggcc 37800 agttaacaaa ttctgatacg gaggacttga ccctatgaac aatgcccatc tctaatctgt 37860 atgagtgcac attcttttta aaaaatggat cttttctact cgtattaata taaaatacat 37920 gcttgtagta aagcaataga gatgaataca aaaaaagttt cccctccagc cttattggtc 37980 tgaggtggtt actactaatg gttgtggatg ttttcacagt catatcagca agtctgctgt 38040 gtgtagggtg gttgtgctct agtcttcctt tcatgatgat gtgtattaaa ttgtttctct 38100 gggagcaata ctcctcgccc ttttaaaaca aatggtaaca gacaccattc cgcacatagc 38160 atattttcat ttagcatctt tggtattaac cagtgtgagc ttttattttt aaagtaacag 38220 tagtgagttg gtggaagtat gatgggttcc tgaagtagtt aaacatggag gcaaaactaa 38280 gtaaactcac cagcttgtta atgtgtggcc acggttgggc acagctccaa tatagggtga 38340 ccagttgacc tggttgctta gaacttgaag gttttccagg aagcagatct tgctttcttc 38400 ttcttcttct tcttcttctt tttttttttt tttgagatgg agtttcattc ttgttgccca 38460 agctggagtg caatgacaca atctcggctc actgcaacct ctacctccta ggttcaagtg 38520 attctcctgc ctcagcctcc caagtagctg ggattacaag tgtacgccac cacacctggc 38580 taattttttg tatttttaga agaaatgggg tttcaccata ttagccaggc tggtctcaaa 38640 ctcctgacct caggtgatcc acccgcctat gcctcccaaa gtgctgggat tacaggcgtg 38700 agccactgcc ccagccagta gagctttgaa accagaacag ttggtcaccc ccaatgtggg 38760 ttctgtgcct ttcccaccct tatcaaccag acaaaatcca ggctctgctt tggctctcag 38820 ctgggatctt gggggtgggg aagtggtaac tgggcgaatt tactcagacc tgagctctaa 38880 gactacctgc tgcctggctt gcagtcatgc atccacagga accagtcttt gcattaagtc 38940 tataactgag acccccagct cccaaggttc caaggtttgt gctacatgct ctcgtttgtg 39000 aggactccaa ggcttcttaa gtctcaaggc aaaggacagg aaggcaaaga tctaagaagg 39060 agaacagagg gttaaattta ttagaatagg aagaaagggg tgaagcaagg tgggtgtgtg 39120 ttgaggggtg agaggttcta gcaagagggc ttaggtctta gccagggtgt atgtagtaag 39180 gggctgctgc catctgggag agcacagcaa gtgaagtggg caagaagaga tgttgccatg 39240 gtatttgagt aacttccact gggtaagtaa gccaccacca ggcaccagct gagctgagtg 39300 gtattcagca gctacttact gtgcacctgt tatgtgccag gcattaggca caagcggtgt 39360 acaagacaaa gtgcccttgt gcagtgaaca cccaagagag acaaatataa aactccaata 39420 ggaataatgt actatgaaga aaaagcaaga caggaggcca gagggtaaaa ggaggtaccc 39480 ttctagccaa aacttccaca tactgttgtg cacattgctt actgcccaac tcaagtttgt 39540 gaattttgac aatttttttg gctgatggca atcgtatgtc ttgatgtgat ggctgttaaa 39600 tttttaaaat aagttttaat gtaaatttaa cgtactattt aggctggtgg tctctgagga 39660 ggtgacactg agctgagacc taaaggatga gaaggagcta ttccttctca ccaatctgca 39720 ttagatgctg tttatcacaa ggtgaaaaat cacttgtttt gaaagagaat gcggaagtgt 39780 agtttgtaga aaagcactga ccatgacctg ataacattcc tagaaatttc cgccatctgg 39840 ggcagcggtg gggaagctgc tgtttgagag actgctgtag tccaggctgt gctgttctga 39900 ccagagcctg ctgcctacag ctgagtggca ggatgtggct agaggaatgg acacagctgt 39960 actggtggga ctcacacaaa tgtcagatgg accagttact 40000 19 20 DNA Artificial Sequence Antisense Oligonucleotide 19 accaaggcag tgagaggaag 20 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 caccagtctt gcccattgat 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21 ctcgctgagg ctccagagct 20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 ctctcaccgc agtactcggc 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23 ctcgtgtact tctggcggct 20 24 20 DNA Artificial Sequence Antisense Oligonucleotide 24 acacgcacac gccgccgccg 20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 tcccgcgccg ccgcccgcac 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 ctcttgacct gggactcgga 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 ggatttcttt agccttctcg 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28 tcttttgtca ggatttcttt 20 29 20 DNA Artificial Sequence Antisense Oligonucleotide 29 agcagtgtaa ctgtttcaac 20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 aagagctaca agcagtgtaa 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 aacgaacctt aagagctaca 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 tgtgtgatct gtctgctctc 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33 ttccaaacat ttgcatttcc 20 34 20 DNA Artificial Sequence Antisense Oligonucleotide 34 ctgcccatcc accaaggcag 20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 gacagaagat ctgcccatcc 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 agtcacacat tggaccctca 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 gaccacagca agtcacacat 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38 attaaatgtc tcagaaatat 20 39 20 DNA Artificial Sequence Antisense Oligonucleotide 39 cgtgaggcca ttggcatgat 20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 cagttatatc cctccatcac 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 tagtttggag cactgaaaat 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 tgcagcttgg ttaccacaac 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43 gttccatgat tgcagcttgg 20 44 20 DNA Artificial Sequence Antisense Oligonucleotide 44 tcgcctctac gaggtgctgg 20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 cattacagga agtagtctgg 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 tcatggcaat actgtacaag 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 atatggttca tggcaatact 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48 ctgacacttt ggagttactg 20 49 20 DNA Artificial Sequence Antisense Oligonucleotide 49 ctattttctg acactttgga 20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 atggcacatc ttttggtcca 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 tatggcacat cttttggtcc 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 gacaagaggc tttgtatttt 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53 ggctgttgat gacaagaggc 20 54 20 DNA Artificial Sequence Antisense Oligonucleotide 54 aagtggtcac ggctgttgat 20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 ttctaaagtg gtcacggctg 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 gttcattcta aagtggtcac 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 caatgaactg gttcattcta 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58 ttcttgacca acaatgtcgc 20 59 20 DNA Artificial Sequence Antisense Oligonucleotide 59 cagaaactgg tttcttgacc 20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 agcgctatgc cagaaactgg 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 aactacaaat agcgctatgc 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 aagcaaaagt aactacaaat 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63 cttattatct gcagtctctc 20 64 20 DNA Artificial Sequence Antisense Oligonucleotide 64 taatgtttac atcttattat 20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 tctacagtca tgctgagtaa 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 agctccaatg attgtttgct 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 ttcattaagc tccaatgatt 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68 atgttcatta agctccaatg 20 69 20 DNA Artificial Sequence Antisense Oligonucleotide 69 tcaaaacaac tcaccaggtt 20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 acagttctgt tcaaaacaac 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 ccattgatac aattaaaatt 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 aattgtaata tgtgaaatac 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73 gcacaccaac aatgtgacaa 20 74 20 DNA Artificial Sequence Antisense Oligonucleotide 74 aacccacaaa gtgcacacca 20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 gaagaaccca caaagtgcac 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 agacttaaat ctcaagttat 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 aagtgacgtg ctgcaaagtt 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78 agtctttggg ttgcatctgt 20 79 20 DNA Artificial Sequence Antisense Oligonucleotide 79 atgaatagga agctttcaag 20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 acccagtctc ttagttttcc 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 tccaggcgtg agccactgtg 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 gctgtcgctt aggctggagt 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83 gggcgaagtg gctcacgcct 20 84 20 DNA Artificial Sequence Antisense Oligonucleotide 84 agctgagagc agcaagtggc 20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 agcttctttt tatatcagca 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 acacaccaaa accccatctc 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87 ataaaggcta atagaggtga 20 88 20 DNA Artificial Sequence Antisense Oligonucleotide 88 acattcgctt aaaagccaaa 20 89 20 DNA Artificial Sequence Antisense Oligonucleotide 89 ttgacattat caaattgtcc 20 90 20 DNA Artificial Sequence Antisense Oligonucleotide 90 gtgcagtagt atgatcatag 20 91 20 DNA Artificial Sequence Antisense Oligonucleotide 91 aatatgctca ctttgtcctg 20 92 20 DNA Artificial Sequence Antisense Oligonucleotide 92 cagtggtttg ttccttttca 20 93 20 DNA Artificial Sequence Antisense Oligonucleotide 93 tggcccctct ggtgtttctg 20 94 20 DNA Artificial Sequence Antisense Oligonucleotide 94 acatgttaat ggattcattg 20 95 20 DNA Artificial Sequence Antisense Oligonucleotide 95 cgaactcctg atctcaggtg 20 96 20 DNA Artificial Sequence Antisense Oligonucleotide 96 aaataaaagt tggaatctga 20 

What is claimed is:
 1. A compound 8 to 50 nucleobases in length targeted to a nucleic acid molecule encoding Protein Phosphatase 2 catalytic subunit alpha, wherein said compound specifically hybridizes with and inhibits the expression of Protein Phosphatase 2 catalytic subunit alpha.
 2. The compound of claim 1 which is an antisense oligonucleotide.
 3. The compound of claim 2 wherein the antisense oligonucleotide has a sequence comprising SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 65, 66, 67, 68, 69, 70, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 87, 89, 90 or
 92. 4. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified internucleoside linkage.
 5. The compound of claim 4 wherein the modified internucleoside linkage is a phosphorothioate linkage.
 6. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified sugar moiety.
 7. The compound of claim 6 wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 8. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified nucleobase.
 9. The compound of claim 8 wherein the modified nucleobase is a 5-methylcytosine.
 10. The compound of claim 2 wherein the antisense oligonucleotide is a chimeric oligonucleotide.
 11. A compound 8 to 50 nucleobases in length which specifically hybridizes with at least an 8-nucleobase portion of an active site on a nucleic acid molecule encoding Protein Phosphatase 2 catalytic subunit alpha.
 12. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent.
 13. The composition of claim 12 further comprising a colloidal dispersion system.
 14. The composition of claim 12 wherein the compound is an antisense oligonucleotide.
 15. A method of inhibiting the expression of Protein Phosphatase 2 catalytic subunit alpha in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of Protein Phosphatase 2 catalytic subunit alpha is inhibited.
 16. A method of treating an animal having a disease or condition associated with Protein Phosphatase 2 catalytic subunit alpha comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of Protein Phosphatase 2 catalytic subunit alpha is inhibited.
 17. The method of claim 16 wherein the disease or condition is a hyperproliferative disorder.
 18. The method of claim 17 wherein the hyperproiferative disorder is cancer.
 19. The method of claim 16 wherein the disease or condition involves aberrant insulin signaling.
 20. The method of claim 16 wherein the disease or condition is diabetes. 